Lubert Stryer

All Publications

• Celebrating the Scientific Life of Richard A. Mathies JOURNAL OF PHYSICAL CHEMISTRY B Stryer, L. 2012; 116 (35): 10409-10410

  View details for DOI 10.1021/jp306719z

  View details for Web of Science ID 000308339400001

  View details for PubMedID 22950706

• Exploring Light and Life Journal of Biological Chemistry Stryer, L. 2012; 287 (19): 15164
• Molecular structure of membrane-targeting calcium sensors in vision: Recoverin and guanylate cyclase-activating protein 2 VERTEBRATE PHOTOTRANSDUCTION AND THE VISUAL CYCLE, PT B Ames, J. B., Ikura, M., Stryer, L. 2000; 316: 121-132

  View details for Web of Science ID 000087601700008

  View details for PubMedID 10800672

• Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases JOURNAL OF BIOLOGICAL CHEMISTRY Ames, J. B., Dizhoor, A. M., Ikura, M., Palczewski, K., Stryer, L. 1999; 274 (27): 19329-19337

  Abstract

  Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nM) and inhibits them at high Ca2+ (>500 nM). The light-induced lowering of the Ca2+ level from approximately 500 nM in the dark to approximately 50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 A in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.

  View details for Web of Science ID 000081196300067

  View details for PubMedID 10383444

• Differential isotope labeling strategy for determining the structure of myristoylated recoverin by NMR spectroscopy JOURNAL OF BIOMOLECULAR NMR Tanaka, T., Ames, J. B., Kainosho, M., Stryer, L., Ikura, M. 1998; 11 (2): 135-152

  Abstract

  The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca(2+)-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with C and the protein was unlabeled or vice versa; in others, both were C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of beta-methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the average minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 +/- 0.07 and 0.55 +/- 0.18 A for backbone atoms, and the 1.09 +/- 0.07 and 1.10 +/- 0.15 A for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 A. Hydrophobic residues Leu28, Trp31, and Tyr32 from a cluster that interacts with the front end of the myristoyl (C1-C8), whereas residues Phe49, Phe56, Tyr86, Val87, and Leu90 interact with the tail end (C9-C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, C14:2 chains.

  View details for Web of Science ID 000074811200003

  View details for PubMedID 9679292

• Molecular mechanics of calcium-myristoyl switches NATURE Ames, J. B., Ishima, R., Tanaka, T., Gordon, J. I., Stryer, L., Ikura, M. 1997; 389 (6647): 198-202

  Abstract

  Many eukaryotic cellular and viral proteins have a covalently attached myristoyl group at the amino terminus. One such protein is recoverin, a calcium sensor in retinal rod cells, which controls the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase. Recoverin has a relative molecular mass of 23,000 (M[r] 23K), and contains an amino-terminal myristoyl group (or related acyl group) and four EF hands. The binding of two Ca2+ ions to recoverin leads to its translocation from the cytosol to the disc membrane. In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF hands. We have used nuclear magnetic resonance to show that Ca2+ induces the unclamping and extrusion of the myristoyl group, enabling it to interact with a lipid bilayer membrane. The transition is also accompanied by a 45-degree rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. The conservation of the myristoyl binding site and two swivels in recoverin homologues from yeast to humans indicates that calcium-myristoyl switches are ancient devices for controlling calcium-sensitive processes.

  View details for Web of Science ID A1997XV75700053

  View details for PubMedID 9296500

• Vision: From photon to perception PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stryer, L. 1996; 93 (2): 557-559

  View details for Web of Science ID A1996TR32600005

  View details for PubMedID 9254392

• Nuclear magnetic resonance evidence for Ca2+-induced extrusion of the myristoyl group of recoverin JOURNAL OF BIOLOGICAL CHEMISTRY Ames, J. B., Tanaka, T., Ikura, M., Stryer, L. 1995; 270 (52): 30909-30913

  Abstract

  Recoverin, a recently discovered member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to translocate to retinal disc membranes when the Ca2+ level is elevated. Two-dimensional 1H-13C shift correlation NMR spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe the effect of Ca2+ on the environment of the attached myristoyl group. In the Ca(2+)-free state, each pair of methylene protons bonded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives rise to two peaks. The splittings, caused by nonequivalent methylene proton chemical shifts, indicate that the myristoyl group interacts intimately with the protein in the Ca(2+)-free state. By contrast, only one peak is seen for each pair of methylene protons in the Ca(2+)-bound state, indicating that the myristoyl group is located in an isotropic environment in this form. Furthermore, the 1H-13C shift correlation NMR spectrum of Ca(2+)-bound recoverin is very similar to that of myristic acid in solution. 1H-(13)C shift correlation NMR experiments were also performed with 13C-labeled recoverin to selectively probe the resonances of methyl groups in the hydrophobic core of the protein. The spectrum of Ca(2+)-bound myristoylated recoverin is different from that of Ca(2+)-free myristoylated recoverin but similar to that of Ca(2+)-bound unmyristoylated recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated recoverin in the Ca(2+)-bound state. Three-dimensional (13C/F1)-edited (13C/F3)-filtered heteronuclear multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe protein residues located within 5 A of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca(2+)-free recoverin, whereas the myristoyl group makes no observable contacts with the protein in the Ca(2+)-bound state. These NMR data demonstrate that the binding of Ca2+ to recoverin induces the extrusion of its myristoyl group into the solvent, which would enable it to interact with a lipid bilayer or a hydrophobic site of a target protein.

  View details for Web of Science ID A1995TN44400013

  View details for PubMedID 8537345

• SEQUESTRATION OF THE MEMBRANE-TARGETING MYRISTOYL GROUP OF RECOVERIN IN THE CALCIUM-FREE STATE NATURE Tanaka, T., Ames, J. B., HARVEY, T. S., Stryer, L., Ikura, M. 1995; 376 (6539): 444-447

  Abstract

  Recoverin, a retinal calcium-binding protein of relative molecular mass (M(r)) 23K, participates in the recovery phase of visual excitation and in adaptation to background light. The Ca(2+)-bound form of recoverin prolongs the photoresponse, probably by blocking phosphorylation of photoexcited rhodopsin. Retinal recoverin contains a covalently attached myristoyl group or related acyl group at its amino terminus and two Ca(2+)-binding sites. Ca2+ binding to myristoylated, but not unmyristoylated, recoverin induces its translocation to bilayer membranes, indicating that the myristoyl group is essential to the read-out of calcium signals (calcium-myristoyl switch). Here we present the solution structure of Ca(2+)-free, myristoylated recombinant recoverin obtained by heteronuclear multidimensional NMR spectroscopy. The myristoyl group is sequestered in a deep hydrophobic pocket formed by many aromatic and other hydrophobic residues from five flanking helices.

  View details for Web of Science ID A1995RM63900058

  View details for PubMedID 7630423

• AMINO-TERMINAL MYRISTOYLATION INDUCES COOPERATIVE CALCIUM-BINDING TO RECOVERIN JOURNAL OF BIOLOGICAL CHEMISTRY Ames, J. B., PORUMB, T., Tanaka, T., Ikura, M., Stryer, L. 1995; 270 (9): 4526-4533

  Abstract

  Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to bind to disc membranes when the Ca2+ level is elevated. Ca(2+)-bound recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation. We report here Ca2+ binding studies of myristoylated and unmyristoylated recombinant recoverin using flow dialysis, fluorescence, and NMR spectroscopy. Unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ with dissociation constants of 0.11 and 6.9 microM. In contrast, two Ca2+ bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 microM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding. One-dimensional 1H and two-dimensional 15N-1H shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca2+ concentration show that a concerted conformational change occurs when two Ca2+ are bound. The Ca2+ binding and NMR data can be fit to a concerted allosteric model in which the two Ca2+ binding sites have different affinities in both the T and R states. The T and R conformational states are defined in terms of the Ca(2+)-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded. Ca2+ binds to the R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 microM, as in unmyristoylated recoverin. The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and < 0.05 for unmyristoylated recoverin. Thus, the attached myristoyl group has two related roles: it shifts the T/R ratio of the unliganded protein more than 8000-fold, and serves as a membrane anchor for the fully liganded protein.

  View details for Web of Science ID A1995QK08400054

  View details for PubMedID 7876221

• EXPRESSION AND CHARACTERIZATION OF CALCIUM-MYRISTOYL SWITCH PROTEINS LIPID MODIFICATIONS OF PROTEINS Zozulya, S., Ladant, D., Stryer, L. 1995; 250: 383-393

  View details for Web of Science ID A1995BD46Q00030

  View details for PubMedID 7651166

• DUAL ROLE OF CALMODULIN IN AUTOPHOSPHORYLATION OF MULTIFUNCTIONAL CAM KINASE MAY UNDERLIE DECODING OF CALCIUM SIGNALS NEURON Hanson, P. I., Meyer, T., Stryer, L., Schulman, H. 1994; 12 (5): 943-956

  Abstract

  Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase makes it Ca2+ independent by trapping bound calmodulin and by enabling the kinase to remain partially active even after calmodulin dissociates. We show that autophosphorylation is an intersubunit reaction between neighbors in the multimeric kinase which requires two molecules of calmodulin. Ca2+/calmodulin acts not only to activate the "kinase" subunit but also to present effectively the "substrate" subunit for autophosphorylation. Conversion of the kinase to the potentiated or trapped state is a cooperative process that is inefficient at low occupancy of calmodulin. Simulations show that repetitive Ca2+ pulses at limiting calmodulin lead to the recruitment of calmodulin to the holoenzyme, which further stimulates autophosphorylation and trapping. This cooperative, positive feedback loop will potentiate the response of the kinase to sequential Ca2+ transients and establish a threshold frequency at which the enzyme becomes highly active.

  View details for Web of Science ID A1994NM83100002

  View details for PubMedID 8185953

• 3-DIMENSIONAL STRUCTURE OF RECOVERIN, A CALCIUM SENSOR IN VISION CELL Flaherty, K. M., Zozulya, S., Stryer, L., McKay, D. B. 1993; 75 (4): 709-716

  Abstract

  Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision. We report here the crystal structure of recombinant unmyristoylated recoverin at 1.9 A resolution. The four EF hands of the protein are arranged in a compact array that contrasts with the dumbbell shape of calmodulin and troponin C. A calcium ion is bound to EF hand 3, while EF hand 2 can bind samarium but not calcium in this crystal form. The other two EF hands have novel structural features that prevent or impair calcium binding. A concave hydrophobic surface formed by EF hands 1 and 2 may participate in the read out of calcium signals by recoverin and its homologs.

  View details for Web of Science ID A1993MH74900016

  View details for PubMedID 8242744

• INTERACTIONS BETWEEN DIVALENT-CATIONS AND THE GATING MACHINERY OF CYCLIC GMP-ACTIVATED CHANNELS IN SALAMANDER RETINAL RODS JOURNAL OF GENERAL PHYSIOLOGY Karpen, J. W., Brown, R. L., Stryer, L., Baylor, D. A. 1993; 101 (1): 1-25

  Abstract

  The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.

  View details for Web of Science ID A1993KG51600001

  View details for PubMedID 7679715

• MOLECULAR MECHANISM OF VISUAL EXCITATION HARVEY LECTURES, VOL 87 Stryer, L. 1993; 87: 129-143

  View details for Web of Science ID A1993BB86V00008

• RANGE OF MESSENGER ACTION OF CALCIUM-ION AND INOSITOL 1,4,5-TRISPHOSPHATE SCIENCE Allbritton, N. L., Meyer, T., Stryer, L. 1992; 258 (5089): 1812-1815

  Abstract

  The range of messenger action of a point source of Ca2+ or inositol 1,4,5-trisphosphate (IP3) was determined from measurements of their diffusion coefficients in a cytosolic extract from Xenopus laevis oocytes. The diffusion coefficient (D) of [3H]IP3 injected into an extract was 283 microns 2/s. D for Ca2+ increased from 13 to 65 microns 2/s when the free calcium concentration was raised from about 90 nM to 1 microM. The slow diffusion of Ca2+ in the physiologic concentration range results from its binding to slowly mobile or immobile buffers. The calculated effective ranges of free Ca2+ before it is buffered, buffered Ca2+, and IP3 determined from their diffusion coefficients and lifetimes were 0.1 micron, 5 microns, and 24 microns, respectively. Thus, for a transient point source of messenger in cells smaller than 20 microns, IP3 is a global messenger, whereas Ca2+ acts in restricted domains.

  View details for Web of Science ID A1992KB96400044

  View details for PubMedID 1465619

• CALCIUM MYRISTOYL PROTEIN SWITCH PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zozulya, S., Stryer, L. 1992; 89 (23): 11569-11573

  Abstract

  Recoverin, a recently discovered member of the EF-hand superfamily of Ca(2+)-binding proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyristoylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. Binding was half-maximal at 1.1 and 1.0 microM Ca2+, respectively. The Hill coefficients of 1.8 and 1.7, respectively, indicate that binding was cooperative. In contrast, Ca2+ induced the binding of myristoylated but not of unmyristoylated recoverin to rod outer segment membranes. Binding to these membranes was half-maximal at 2.1 microM Ca2+, and the Hill coefficient was 2.4. Likewise, myristoylated but not unmyristoylated recoverin exhibited Ca(2+)-induced binding to phosphatidylcholine vesicles. These findings suggest that the binding of Ca2+ to recoverin has two effects: (i) hydrophobic surfaces are exposed, allowing the protein to interact with complementary nonpolar sites, such as the aromatic rings of phenyl-agarose; and (ii) the myristoyl group is extruded, enabling recoverin to insert into a lipid bilayer membrane. The myristoyl group is likely to be an active participant in Ca2+ signaling by recoverin and related EF-hand proteins such as visinin and neurocalcin.

  View details for Web of Science ID A1992KA90300098

  View details for PubMedID 1454850

• CLONING, EXPRESSION, AND CRYSTALLIZATION OF RECOVERIN, A CALCIUM SENSOR IN VISION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ray, S., Zozulya, S., Niemi, G. A., Flaherty, K. M., BROLLEY, D., Dizhoor, A. M., McKay, D. B., Hurley, J., Stryer, L. 1992; 89 (13): 5705-5709

  Abstract

  Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

  View details for Web of Science ID A1992JC86800002

  View details for PubMedID 1385864

  View details for PubMedCentralID PMC49365

• CALMODULIN TRAPPING BY CALCIUM-CALMODULIN DEPENDENT PROTEIN-KINASE SCIENCE Meyer, T., Hanson, P. I., Stryer, L., Schulman, H. 1992; 256 (5060): 1199-1202

  Abstract

  Multifunctional calcium-calmodulin-dependent protein kinase (CaM kinase) transduces transient elevations in intracellular calcium into changes in the phosphorylation state and activity of target proteins. By fluorescence emission anisotropy, the affinity of CaM kinase for dansylated calmodulin was measured and found to increase 1000 times after autophosphorylation of the threonine at position 286 of the protein. Autophosphorylation markedly slowed the release of bound calcium-calmodulin; the release time increased from less than a second to several hundred seconds. In essence, calmodulin is trapped by autophosphorylation. The shift in affinity does not occur in a site-directed mutant in which threonine at position 286 has been replaced by a non-phosphorylatable amino acid. These experiments demonstrate the existence of a new state in which calmodulin is bound to CaM kinase even though the concentration of calcium is basal. Calmodulin trapping provides for molecular potentiation of calcium transients and may enable detection of their frequency.

  View details for Web of Science ID A1992HV19200035

  View details for PubMedID 1317063

• VISUAL EXCITATION AND RECOVERY JOURNAL OF BIOLOGICAL CHEMISTRY Stryer, L. 1991; 266 (17): 10711-10714

  View details for Web of Science ID A1991FQ77400001

  View details for PubMedID 1710212

• CALCIUM SPIKING ANNUAL REVIEW OF BIOPHYSICS AND BIOPHYSICAL CHEMISTRY Meyer, T., Stryer, L. 1991; 20: 153-174

  View details for Web of Science ID A1991FQ36000007

  View details for PubMedID 1867714

• Molecular mechanism of visual excitation. Harvey lectures Stryer, L. 1991; 87: 129-143

  View details for PubMedID 1688322

• OPTIMIZATION OF HIGH-SENSITIVITY FLUORESCENCE DETECTION ANALYTICAL CHEMISTRY Mathies, R. A., Peck, K., Stryer, L. 1990; 62 (17): 1786-1791

  Abstract

  We present general expressions for the number of photons emitted by a fluorescent chromophore as a function of the intensity and the duration of illumination. The aim is to find optimal conditions for detecting fluorescent molecules in the presence of both ground-state depletion and photodestruction. The key molecular parameters are the absorption coefficient epsilon, the excited singlet-state lifetime tau f, the excited triplet-state decay rate kT, the intersystem crossing rate kI, and the intrinsic photodestruction time tau d. When only singlet saturation and photochemistry are important, the signal-to-noise ratio depends on two fundamental variables: k, the ratio of the absorption rate ka to the observed fluorescence decay rate kf, and tau, the ratio of the duration of illumination taut to the intrinsic photodestruction time tau d. Equations are also developed for the more complicated cases when triplet formation and photochemistry are important. This theory was tested by measuring the fluorescence from a solution of beta-phycoerythrin flowed through a focused argon ion laser beam. The dependence of the fluorescence on the incident light intensity and the illumination time agrees well with the theoretical prediction for singlet saturation and photochemistry. The signal-to-noise ratio is optimal when the light intensity and the flow rate are adjusted so that both K and tau are close to unity (5 X 10(22) photons cm-2 s-1 and a transit time tau t of 700 mus). This analysis should be useful for optimizing fluorescence detection in DNA sequencing, chromatography, fluorescence microscopy, and single-molecular fluorescence detection.

  View details for Web of Science ID A1990DW85300014

  View details for PubMedID 2240569

• TRANSIENT CALCIUM RELEASE INDUCED BY SUCCESSIVE INCREMENTS OF INOSITOL 1,4,5-TRISPHOSPHATE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Meyer, T., Stryer, L. 1990; 87 (10): 3841-3845

  Abstract

  Many hormonal, neurotransmitter, and sensory stimuli trigger the formation of inositol 1,4,5-trisphosphate, which in turn releases calcium from intracellular stores. We report here that inositol 1,4,5-trisphosphate-induced calcium release from saponin-permeabilized rat basophilic leukemia cells at 37 degrees C is markedly biphasic, in contrast with nearly monophasic release kinetics at 11 degrees C. Hepatoma, PC-12 neuronal cells, and several other cell types exhibit similar biphasic release at 37 degrees C. The biphasic kinetics are not due to degradation of inositol 1,4,5-trisphosphate or to increased Ca2(+)-ATPase pump activity. Biphasic calcium release was also seen when ATP was quenched to less than 0.4 microM by adding hexokinase and glucose, suggesting that phosphorylation is not involved. External calcium (100 nM-600 nM) range had little influence on the biphasic kinetics. Rapid-mixing experiments revealed that rapid efflux of calcium is followed in approximately 0.5 s by a 30-fold slower efflux. Most striking, successive additions of the same amount of inositol 1,4,5-trisphosphate induced short bursts of calcium release of similar size. This retention of responsiveness, which we term increment detection, may be a distinct mode of signal transduction. Like inactivation and adaptation, increment detection gives rise to transient responses to sustained stimuli. Systems exhibiting inactivation, adaptation, and increment detection differ in their responsiveness (none, partial, and full, respectively) to stepwise increases in stimulus intensity. Increment detection could be advantageous in generating receptor-triggered calcium oscillations.

  View details for Web of Science ID A1990DD87300043

  View details for PubMedID 2339124

• ACTIVATION MECHANISM OF RETINAL ROD CYCLIC-GMP PHOSPHODIESTERASE PROBED BY FLUORESCEIN-LABELED INHIBITORY SUBUNIT BIOCHEMISTRY Wensel, T. G., Stryer, L. 1990; 29 (8): 2155-2161

  Abstract

  The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is alpha beta gamma 2. T alpha-GTP and gamma act reciprocally. We have investigated the activation mechanism using fluorescein-labeled gamma subunit (gamma F) as a probe. gamma F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). gamma F, like native gamma, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by gamma F was overcome by further addition of T alpha-GTP. gamma F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. gamma F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that gamma F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 approximately 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of gamma from the inhibitory sites. The binding of gamma F to trypsin-activated PDE alpha beta was monitored by steady-state emission anisotropy measurements and compared with PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1990CQ48200028

  View details for PubMedID 2158346

• KINETICS OF CALCIUM-CHANNEL OPENING BY INOSITOL 1,4,5-TRISPHOSPHATE BIOCHEMISTRY Meyer, T., Wensel, T., Stryer, L. 1990; 29 (1): 32-37

  Abstract

  The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.

  View details for Web of Science ID A1990CH52900004

  View details for PubMedID 1691015

• PHYCOBILIPROTEIN AVIDIN AND PHYCOBILIPROTEIN BIOTIN CONJUGATES METHODS IN ENZYMOLOGY Glazer, A. N., Stryer, L. 1990; 184: 188-194

  View details for Web of Science ID A1990MC41800020

  View details for PubMedID 2388569

• EXPRESSION IN BACTERIA OF FUNCTIONAL INHIBITORY SUBUNIT OF RETINAL ROD CGMP PHOSPHODIESTERASE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brown, R. L., Stryer, L. 1989; 86 (13): 4922-4926

  Abstract

  The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory gamma subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine gamma subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of lambda phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the lambda cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue gamma subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of gamma in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic gamma with the same amino acid sequence as that of native gamma. Both fusion protein and synthetic gamma inhibited trypsin-activated phosphodiesterase with high affinity (Kd less than 100 pM). Likewise, both were as effective as native gamma in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of gamma is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of gamma stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of gamma is critical for inhibition of the phosphodiesterase.

  View details for Web of Science ID A1989AE15800025

  View details for PubMedID 2544882

• SINGLE-MOLECULE FLUORESCENCE DETECTION - AUTO-CORRELATION CRITERION AND EXPERIMENTAL REALIZATION WITH PHYCOERYTHRIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peck, K., Stryer, L., Glazer, A. N., Mathies, R. A. 1989; 86 (11): 4087-4091

  Abstract

  A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The autocorrelation function reveals single-molecule events and provides a criterion for optimizing experimental parameters. The transit time of fluorescent molecules through the 120-fl imaged volume was 800 microseconds. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 x 10(23) photons.cm-2.s-1, corresponding to a mean time of 1.1 ns between absorptions. The mean incremental count rate was 1.5 per 100 microseconds for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was achieved. At this concentration, the mean occupancy was 0.014 monomer molecules in the probed volume. A hard-wired version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule counter is 3 orders of magnitude more sensitive than conventional fluorescence detection systems.

  View details for Web of Science ID A1989U940700035

  View details for PubMedID 2726766

• STEREOCHEMICAL COURSE OF THE REACTION CATALYZED BY THE CYCLIC-GMP PHOSPHODIESTERASE FROM RETINAL ROD OUTER SEGMENTS JOURNAL OF BIOLOGICAL CHEMISTRY Eckstein, F., Karpen, J. W., CRITCHFIELD, J. M., Stryer, L. 1988; 263 (28): 14080-14085

  Abstract

  The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.

  View details for Web of Science ID A1988Q306000019

  View details for PubMedID 2844755

• STATISTICAL DISTRIBUTIONS OF NUCLEOSOMES - NONRANDOM LOCATIONS BY A STOCHASTIC MECHANISM NUCLEIC ACIDS RESEARCH Kornberg, R. D., Stryer, L. 1988; 16 (14): 6677-6690

  View details for Web of Science ID A1988P435100030

• Statistical distributions of nucleosomes: nonrandom locations by a stochastic mechanism. Nucleic acids research Kornberg, R. D., Stryer, L. 1988; 16 (14A): 6677-6690

  Abstract

  Expressions are derived for distributions of nucleosomes in chromatin. Nucleosomes are placed on DNA at the densities found in bulk chromatin, and their locations are allowed to vary at random. No further assumptions are required to simulate the periodic patterns of digestion obtained with various nucleases. The introduction of a boundary constraint, due for example to sequence-specific protein binding, results in an array of regularly spaced nucleosomes at nonrandom locations, similar to the arrays reported for some genes and other chromosomal regions.

  View details for PubMedID 3399412

• HIGHLY COOPERATIVE FEEDBACK-CONTROL OF RETINAL ROD GUANYLATE-CYCLASE BY CALCIUM-IONS NATURE Koch, K. W., Stryer, L. 1988; 334 (6177): 64-66

  Abstract

  Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.

  View details for Web of Science ID A1988P120900056

  View details for PubMedID 2455233

• MOLECULAR-MODEL FOR RECEPTOR-STIMULATED CALCIUM SPIKING PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Meyer, T., Stryer, L. 1988; 85 (14): 5051-5055

  Abstract

  Many cells exhibit periodic transient increases in cytosolic calcium levels rather than a sustained rise when stimulated by a hormone or growth factor. We propose here a molecular model that accounts for periodic calcium spiking induced by a constant stimulus. Four elements give rise to repetitive calcium transients: cooperativity and positive feedback between a pair of reciprocally coupled (crosscoupled) messengers, followed by deactivation and then by reactivation. The crosscoupled messengers in our model are inositol 1,4,5-trisphosphate (InsP3) and cytosolic calcium ions. The opening of calcium channels in the endoplasmic reticulum by the binding of multiple molecules of InsP3 provides the required cooperativity. The stimulation of receptor-activated phospholipase C by released calcium ions leads to positive feedback. InsP3 is destroyed by a phosphatase, and calcium ion is pumped back into the endoplasmic reticulum. These processes generate bistability: the cytosolic calcium concentration abruptly increases from a basal level to a stimulated level at a threshold degree of activation of phospholipase C. Spiking further requires slow deactivation and subsequent reactivation. In our model, mitochondrial sequestration of calcium ion prevents the cytosolic level from increasing above several micromolar and enables the system to return to the basal state. When the endoplasmic reticulum calcium store is refilled to a critical level by the Ca2+-ATPase pump, cooperative positive feedback between the InsP3-gated channel and phospholipase C begins again to give the next calcium spike. The time required for the calcium level in the endoplasmic reticulum to reach a threshold sets the interval between spikes. The amplitude, shape, and period of calcium spikes calculated for this model are like those observed experimentally.

  View details for Web of Science ID A1988P362000022

  View details for PubMedID 2455890

• SEGMENTAL FLEXIBILITY AND COMPLEMENT-FIXATION OF GENETICALLY ENGINEERED CHIMERIC HUMAN, RABBIT AND MOUSE ANTIBODIES EMBO JOURNAL Dangl, J. L., Wensel, T. G., Morrison, S. L., Stryer, L., Herzenberg, L. A., Oi, V. T. 1988; 7 (7): 1989-1994

  Abstract

  We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen-combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

  View details for Web of Science ID A1988P074100009

  View details for PubMedID 3138110

• HIGHLY COOPERATIVE OPENING OF CALCIUM CHANNELS BY INOSITOL 1,4,5-TRISPHOSPHATE SCIENCE Meyer, T., Holowka, D., Stryer, L. 1988; 240 (4852): 653-656

  Abstract

  The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.

  View details for Web of Science ID A1988N126500033

  View details for PubMedID 2452482

• GENETICALLY ENGINEERED IMMUNOGLOBULINS REVEAL STRUCTURAL FEATURES CONTROLLING SEGMENTAL FLEXIBILITY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schneider, W. P., Wensel, T. G., Stryer, L., Oi, V. T. 1988; 85 (8): 2509-2513

  Abstract

  We have carried out nanosecond fluorescence polarization studies of genetically engineered immunoglobulins to determine the structural features controlling their segmental flexibility. The proteins studied were hybrids of a relatively rigid isotype (mouse IgG1) and a relatively flexible one (mouse IgG2a). They have identical light chains and heavy chain variable regions and have the same combining sites for epsilon-dansyl-L-lysine, a fluorescent hapten. The fluorescence of the bound dansyl chromophore was excited at 348 nm with subnanosecond laser pulses, and the emission in the nanosecond time range was measured with a single-photon-counting apparatus. The emission anisotropy kinetics of the hybrid antibodies revealed that segmental flexibility is controlled by the heavy chain constant region 1 (CH1) as well as by the hinge. In contrast, the CH2 and CH3 domains did not influence segmental flexibility. The hinge and CH1 domains must be properly matched to allow facile movement of the Fab units. Studies of hybrids of IgG1 and IgG2a within CH1 showed that the loop formed by residues 131-139 is important in controlling segmental flexibility. X-ray crystallographic studies by others of human IgG1 have shown that this loop makes several van der Waals contacts with the hinge.

  View details for Web of Science ID A1988N023400020

  View details for PubMedID 3128789

• GATING KINETICS OF THE CYCLIC-GMP-ACTIVATED CHANNEL OF RETINAL RODS - FLASH-PHOTOLYSIS AND VOLTAGE-JUMP STUDIES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Karpen, J. W., Zimmerman, A. L., Stryer, L., Baylor, D. A. 1988; 85 (4): 1287-1291

  Abstract

  The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester ("caged" cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (greater than 50 microM), whereas at physiological cGMP concentrations (less than 5 microM), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.

  View details for Web of Science ID A1988M196200071

  View details for PubMedID 2448798

• MOLECULAR-BASIS OF VISUAL EXCITATION COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Stryer, L. 1988; 53: 283-294

  View details for Web of Science ID A1988AP53800034

  View details for PubMedID 3076083

• MOLECULAR MECHANICS OF THE CYCLIC-GMP-ACTIVATED CHANNEL OF RETINAL RODS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Karpen, J. W., Zimmerman, A. L., Stryer, L., Baylor, D. A. 1988; 53: 325-332

  View details for Web of Science ID A1988AP53800038

  View details for PubMedID 2855483

• VISUAL TRANSDUCTION - DESIGN AND RECURRING MOTIFS CHEMICA SCRIPTA Stryer, L. 1987; 27B: 161-171

  View details for Web of Science ID A1987L820300020

• THE MOLECULES OF VISUAL EXCITATION SCIENTIFIC AMERICAN Stryer, L. 1987; 257 (1): 42-?

  View details for Web of Science ID A1987H745500002

  View details for PubMedID 3037690

• PHOTOSTABILITY STUDIES OF PHYCOBILIPROTEIN FLUORESCENT LABELS ANALYTICAL BIOCHEMISTRY White, J. C., Stryer, L. 1987; 161 (2): 442-452

  Abstract

  Photostability studies of four fluorescent phycobiliproteins were conducted to identify stable chromophores for biological labeling applications. Phycobiliprotein photodestruction was linear with the applied laser power and depended on the total number of photons absorbed per molecule. Photodestruction quantum yields phi of 1.1 X 10(-5) for R-phycoerythrin, 6.6 X 10(-6) for B-phycoerythrin, 4.5 X 10(-6) for allophycocyanin, and 2.5 X 10(-6) for C-phycocyanin were measured. C-Phycocyanin is a factor of 10.8 more photostable than fluorescein. The photostability of R-phycoerythrin was improved by a factor of 1.7 by adding n-propyl gallate. The addition of superoxide dismutase, catalase, sodium dithionite, ascorbate, dithiothreitol, EDTA, or deoxygenation with argon bubbling had no effect on the photostability of R-phycoerythrin.

  View details for Web of Science ID A1987G324900034

  View details for PubMedID 3578804

• INTERACTION OF RETINAL TRANSDUCIN WITH GUANOSINE TRIPHOSPHATE ANALOG - SPECIFICITY OF THE GAMMA-PHOSPHATE BINDING REGION BIOCHEMISTRY Yamanaka, G., Eckstein, F., Stryer, L. 1986; 25 (20): 6149-6153

  Abstract

  The interaction of six hydrolysis-resistant analogues of GTP with transducin, the signal-coupling protein in vertebrate photoreceptors, was investigated. GppNHp and GppCH2p differ from GTP at the bridging position between the beta- and gamma-phosphate groups. The other analogues studied (GTP gamma F, GTP gamma OMe, GTP gamma OPh, and GTP gamma S) differ from GTP in containing a substituent on the gamma-phosphorus atom or at a nonbridging gamma-oxygen atom. Competition binding experiments were carried out by adding an analogue, [alpha-32P]GTP, and a catalytic amount of photoexcited rhodopsin (R) to transducin and measuring the amount of bound [gamma-32P]GTP. The order of effectiveness of these analogues in binding to transducin was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh greater than GTP gamma OMe greater than GppCH2p greater than GTP gamma F A second assay measured the effectiveness of GTP gamma S, GppNHp, and GppCH2p in eluting transducin from disc membranes containing R. The basis of this assay is that transducin is released from disc membranes when it is activated to the GTP form. The relative potency of these three analogues in converting transducin from a membrane-bound to a soluble form was 1000, 75, and 1, respectively. Stimulation of cGMP phosphodiesterase activity served as a third criterion of the interaction of these analogues with transducin. The order of effectiveness of these analogues in promoting the transducin-mediated activation of the phosphodiesterase was GTP gamma S greater than GTP much greater than GppNHp greater than GTP gamma OPh much greater than GppCH2p greater than GTP gamma OMe greater than GTP gamma F GTP gamma S was more than a 1000 times as potent as GTP gamma F in activating the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1986E515900048

  View details for PubMedID 3466646

• COOPERATIVE POLYMERIZATION REACTIONS - ANALYTICAL APPROXIMATIONS, NUMERICAL EXAMPLES, AND EXPERIMENTAL STRATEGY BIOPHYSICAL JOURNAL Goldstein, R. F., Stryer, L. 1986; 50 (4): 583-599

  Abstract

  How does one obtain kinetic rate constants from the time course of a reversible and cooperative polymerization reaction? We examine a simple version of the homogeneous nucleation-elongation model with both analytical and numerical techniques to test some common assumptions and develop an experimental strategy. The assumption of irreversible polymer formation is found to be a useful and adequate approximation for the numerical determination of monomer disappearance. The assumption of early "pre-equilibrium" between monomer and seed, however, is shown numerically and analytically to produce significant errors over a wide range of parameters, particularly for small seed lengths. We exhibit numerical solutions for many different parameters, and also discuss analytical techniques that allow approximate solutions for several conditions: the high-concentration limit; the long-time limit; and the long-seed-length, lows concentration limit. The overall reaction simplifies when the monomer concentration is large. An experimental strategy for elucidating the seed size and the rate constants for polymerization and depolymerization is presented.

  View details for Web of Science ID A1986E309700004

  View details for PubMedID 3779001

  View details for PubMedCentralID PMC1329836

• Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin. Proteins Wensel, T. G., Stryer, L. 1986; 1 (1): 90-99

  Abstract

  The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.

  View details for PubMedID 2835761

• KINSHIP OF CEPHALOPOD PHOTORECEPTOR G-PROTEIN WITH VERTEBRATE TRANSDUCIN FEBS LETTERS Tsuda, M., Tsuda, T., Terayama, Y., Fukada, Y., Akino, T., Yamanaka, G., Stryer, L., Katada, T., Ui, M., Ebrey, T. 1986; 198 (1): 5-10

  View details for Web of Science ID A1986A844000002

• G-PROTEINS - A FAMILY OF SIGNAL TRANSDUCERS ANNUAL REVIEW OF CELL BIOLOGY Stryer, L., Bourne, H. R. 1986; 2: 391-419

  View details for Web of Science ID A1986E986500014

  View details for PubMedID 3103658

• CYCLIC-GMP CASCADE OF VISION ANNUAL REVIEW OF NEUROSCIENCE Stryer, L. 1986; 9: 87-119

  Abstract

  Cyclic GMP is central to visual excitation in vertebrate retinal rod cells. Sodium channels in the plasma membrane of the outer segment are kept open in the dark by a high level of cGMP. Light closes these channels by activating an enzymatic cascade that leads to the rapid hydrolysis of cGMP. Photoexcited rhodopsin triggers transducin by catalyzing the exchange of GTP for bound GDP. The activated GTP-form of transducin then switches on the phosphodiesterase by overcoming an inhibitory constraint. The overall gain of this cascade is about 10(5). The cascade is turned off by the GTPase activity of transducin and by the action of rhodopsin kinase and arrestin. One of the challenges now is to delineate the interplay of cGMP, calcium ion, and phosphoinositides in excitation and adaptation. Transducin belongs to a family of signal-coupling proteins that includes the G proteins of the hormone-regulated adenylate cyclase cascade. The initial events in visual excitation in molluscs and arthropods are probably similar to those of vertebrates. The triggering of transducin by photoexcited rhodopsin is a recurring motif in visual transduction. The coming together of electrophysiology, biochemistry, and molecular genetics affords new opportunities in unraveling the molecular mechanism of visual transduction.

  View details for Web of Science ID A1986A575000005

  View details for PubMedID 2423011

• STEREOCHEMISTRY OF THE GUANYL NUCLEOTIDE BINDING-SITE OF TRANSDUCIN PROBED BY PHOSPHOROTHIOATE ANALOGS OF GTP AND GDP BIOCHEMISTRY Yamanaka, G., Eckstein, F., Stryer, L. 1985; 24 (27): 8094-8101

  Abstract

  The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp)-GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta-phosphorus atom of GTP. These results lead to the following conclusions: The pro-R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1985AXH2500039

  View details for PubMedID 3004574

• INTERACTION OF HYDROLYSIS-RESISTANT ANALOGS OF CYCLIC-GMP WITH THE PHOSPHODIESTERASE AND LIGHT-SENSITIVE CHANNEL OF RETINAL ROD OUTER SEGMENTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zimmerman, A. L., Yamanaka, G., Eckstein, F., Baylor, D. A., Stryer, L. 1985; 82 (24): 8813-8817

  Abstract

  cGMP opens cation-selective channels when applied to the cytoplasmic side of excised patches of membrane from retinal rod outer segments (ROS). If the light-sensitive channel in intact rods is gated only by cGMP, it should be possible to find a hydrolysis-resistant analog of cGMP that blocks the normal response to light by holding the channel open independent of the degree of illumination. We have studied the interaction of 8-bromo-cGMP (8-Br-cGMP) and the SP and RP phosphorothioate derivatives of cGMP [(Sp)-cGMP[S] and (RP)-cGMP[S]) with the cGMP phosphodiesterase (PDEase) of ROS, the cGMP-sensitive channel of excised ROS patches, and the light-sensitive channel of intact rods. All three analogs were hydrolyzed by PDEase much more slowly than was cGMP. The maximal rates of hydrolysis of 8-Br-cGMP, (SP)-cGMP[S], and (RP)-cGMP[S] were 7.3, 3.7, and less than 0.2 s-1, respectively, compared with 4000 s-1 for cGMP. These analogs are effective competitive inhibitors of the PDEase, with Ki values of 48, 25, and 90 microM, respectively. The nucleotide-activated conductances of excised patches were half-maximal at concentrations of 1.6, 210, and 1200 microM, respectively, compared with 17 microM for cGMP. Thus, 8-Br-cGMP is a highly potent channel agonist. The effects of these analogs on the dark current and photoresponses of intact rod cells were also measured. A suction electrode monitored membrane current across the ROS, while a patch electrode sealed on the inner segment was used to introduce a cGMP analog and to control membrane potential. All three analogs increased the dark current and markedly slowed the response to light flashes. 8-Br-cGMP increased the dark current of the outer segment as much as 48-fold. After the concentration of this analog had risen sufficiently, little of the current could be shut off by light, as expected of a direct effect on the light-sensitive channel of the plasma membrane. These results are consistent with the notions that (i) the light-sensitive channel of rods is controlled solely by the instantaneous concentration of cGMP and (ii) the cGMP-sensitive channel of excised patches is identical to the light-sensitive channel of intact rods.

  View details for Web of Science ID A1985AXL4200115

  View details for PubMedID 2417228

• MOLECULAR DESIGN OF AN AMPLIFICATION CASCADE IN VISION BIOPOLYMERS Stryer, L. 1985; 24 (1): 29-47

  View details for Web of Science ID A1985ACK4500005

  View details for PubMedID 2985139

• PHYCOFLUOR PROBES TRENDS IN BIOCHEMICAL SCIENCES Glazer, A. N., Stryer, L. 1984; 9 (10): 423-427

  View details for Web of Science ID A1984TS73700004

• CORRELATION BETWEEN SEGMENTAL FLEXIBILITY AND EFFECTOR FUNCTION OF ANTIBODIES NATURE Oi, V. T., Vuong, T. M., Hardy, R., REIDLER, J., Dangl, J., Herzenberg, L. A., Stryer, L. 1984; 307 (5947): 136-140

  Abstract

  Mouse monoclonal anti-dansyl antibodies with the same antigen-binding sites but different heavy chain constant regions were generated. The extent of segmental flexibility in times of nanoseconds and the capacity to fix complement were greatest for IgG2b, intermediate for IgG2a, and least for IgG1 and IgE. Hence, the effector functions of immunoglobulin isotypes may be controlled in part by the freedom of movement of their Fab arms.

  View details for Web of Science ID A1984RY26500041

  View details for PubMedID 6690993

• MILLISECOND ACTIVATION OF TRANSDUCIN IN THE CYCLIC-NUCLEOTIDE CASCADE OF VISION NATURE Vuong, T. M., Chabre, M., Stryer, L. 1984; 311 (5987): 659-661

  Abstract

  Cyclic GMP has been implicated as a messenger molecule involved in visual transduction. Photoexcited rhodopsin (R*) binds to a multisubunit membrane protein called transducin (T) and stimulates the exchange of a bound GDP molecule for GTP. This leads to the release of the alpha-subunit of T with bound GTP (T alpha-GTP), which activates a cyclic GMP phosphodiesterase. The question arises as to whether the hydrolysis of cyclic GMP that results from activation of the phosphodiesterase is sufficiently rapid to be involved in visual excitation, which occurs on a time scale of approximately 2 s in the single-photon limit. Previous studies have suggested that the cyclic GMP phosphodiesterase is activated in less than 100 ms at moderate light levels. We report here light scattering studies of magnetically orientated frog rod outer segments which show that a molecule of R* catalyses the activation of a molecule of T in about 1 ms. Thus, hundreds of molecules can be activated within the response time of vision in the single-photon limit, and the formation of T alpha-GTP is fast enough for it to be a key step in visual transduction.

  View details for Web of Science ID A1984TN96200050

  View details for PubMedID 6090950

• TRANSDUCIN AND THE CYCLIC-GMP PHOSPHODIESTERASE - AMPLIFIER PROTEINS IN VISION COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Stryer, L. 1983; 48: 841-852

  Abstract

  Our experiments have delineated the flow of information in the cyclic nucleotide cascade of vision of ROS. A single, photoexcited rhodopsin molecule activates several hundred phosphodiesterase molecules in two stages. First, photoexcited rhodopsin (R*) interacts with transducin (T), a peripheral membrane protein consisting of alpha- (39 kD), beta- (36 kD), and gamma- (approximately 10 kD) subunits. R* catalyzes the exchange of GTP for GDP bound to the subunit of transducin. About 500 T alpha- GTPs are produced per photoexcited rhodopsin at low light levels. T alpha-GTP, released from the beta- and gamma-subunits of transducin, then interacts with the phosphodiesterase to relieve the inhibitory constraint imposed by its gamma-subunit. Hydrolysis of GTP bound to T alpha serves to restore the system to the dark state. Transducin is the amplified signal carrier in this light-triggered cascade. The formation of hundreds of T alpha- GTPs is likely to be the first stage of amplification in visual excitation. The photoactivation of the phosphodiesterase in ROS closely resembles the activation of adenylate cyclase in hormone-sensitive cells. Our cholera toxin labeling studies have shown that transducin is akin to the signal-coupling G protein of the adenylate cyclase system. Cholera toxin specifically ADP- ribosylates and inactivates the GTPase activity of T alpha, just as it does with Gs. The action of pertussis toxin on ROS further underscores the homology of the photoreceptor and hormone-responsive systems. It seems likely that transducin, the stimulatory G protein, and the inhibitory G protein are members of the same family of signal-amplifying proteins. The study of the cyclic nucleotide cascade of vision is proving to be rewarding in affording a view of a recurring motif of signal amplification in nature in addition to providing insight into the mechanism of vision.

  View details for Web of Science ID A1983SX49600086

  View details for PubMedID 6327179

• TRANSDUCIN AND THE CYCLIC-GMP PHOSPHODIESTERASE OF RETINAL ROD OUTER SEGMENTS METHODS IN ENZYMOLOGY Stryer, L., Hurley, J. B., Fung, B. K. 1983; 96: 617-627

  View details for Web of Science ID A1983RU27400051

  View details for PubMedID 6318024

• PURIFICATION AND CHARACTERIZATION OF THE GAMMA-REGULATORY SUBUNIT OF THE CYCLIC-GMP PHOSPHODIESTERASE FROM RETINAL ROD OUTER SEGMENTS JOURNAL OF BIOLOGICAL CHEMISTRY Hurley, J. B., Stryer, L. 1982; 257 (18): 1094-1099

  View details for Web of Science ID A1982PH18200090

• FLUORESCENCE ENERGY-TRANSFER MEASUREMENTS OF DISTANCES IN RHODOPSIN AND THE PURPLE MEMBRANE-PROTEIN METHODS IN ENZYMOLOGY Stryer, L., THOMAS, D. D., CARLSEN, W. F. 1982; 81: 668-678

  View details for Web of Science ID A1982NN70500091

  View details for PubMedID 7098907

• TRANSVERSE LOCATION OF THE RETINAL CHROMOPHORE OF RHODOPSIN IN ROD OUTER SEGMENT DISK MEMBRANES JOURNAL OF MOLECULAR BIOLOGY THOMAS, D. D., Stryer, L. 1982; 154 (1): 145-157

  View details for Web of Science ID A1982MY97000010

  View details for PubMedID 7077659

• RAPID MOTIONS IN PROTEIN MOLECULES BIOCHEMICAL SOCIETY SYMPOSIA Stryer, L. 1982: 39-55

  View details for Web of Science ID A1982PJ02000003

• DIFFUSION-ENHANCED FLUORESCENCE ENERGY-TRANSFER ANNUAL REVIEW OF BIOPHYSICS AND BIOENGINEERING Stryer, L., THOMAS, D. D., Meares, C. F. 1982; 11: 203-222

  View details for Web of Science ID A1982NR16400010

  View details for PubMedID 7049062

• ACTIN-FILAMENTS UNDERGO LIMITED SUBUNIT EXCHANGE IN PHYSIOLOGICAL SALT CONDITIONS JOURNAL OF CELL BIOLOGY Pardee, J. D., Simpson, P. A., Stryer, L., Spudich, J. A. 1982; 94 (2): 316-324

  Abstract

  The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (IAENS) served as the fluorescent energy donor, and actin labeled with either fluorescein-5-isothiocyanate (FITC) or fluorescein-5-maleimide (FM) served as the energy acceptor. Fluorescent-labeled actins from Dictyostelium amoebae and rabbit skeletal muscle were very similar to their unlabeled counterparts with respect to critical actin concentration for filament assembly, assembly rate, ATP hydrolysis upon assembly, and steady-state ATPase. As evidenced by two different types of fluorescence energy transfer experiments, less than 5% of the actin filament subunits exchanged under a variety of buffer conditions at actin concentrations greater than 0.5 mg/ml. At all actin concentrations limited exchange to a plateau level occurred with a half-time of about 20 min. Nearly identical results were obtained when exchange was quantitated by incorporation of 35S-labeled Dictyostelium actin monomers into unlabeled muscle actin or Dictyostelium actin filaments. Furthermore, the proportion of filament-bound ADP which exchanged with [14C]-ATP was nearly the same as actin subunit exchange measured by fluorescence energy transfer and 35S-labeled actin incorporation. These experiments demonstrate that under approximately physiologic ionic conditions only a small percentage of subunits in highly purified skeletal muscle or Dictyostelium F-actin participate in exchange.

  View details for Web of Science ID A1982NZ60200009

  View details for PubMedID 7202009

• ACTIN AND MYOSIN - CONTROL OF FILAMENT ASSEMBLY PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES Spudich, J. A., Pardee, J. D., Simpson, P. A., Yamamoto, K., Kuczmarski, E. R., Stryer, L. 1982; 299 (1095): 247-261

  Abstract

  Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.

  View details for Web of Science ID A1982PP66100010

  View details for PubMedID 6129660

• MECHANISM OF INTERACTION OF DICTYOSTELIUM SEVERIN WITH ACTIN-FILAMENTS JOURNAL OF CELL BIOLOGY Yamamoto, K., Pardee, J. D., REIDLER, J., Stryer, L., Spudich, J. A. 1982; 95 (3): 711-719

  Abstract

  Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.

  View details for Web of Science ID A1982PT01600004

  View details for PubMedID 6897549

• CONTROL OF ASSEMBLY OF DICTYOSTELIUM MYOSIN AND ACTIN-FILAMENTS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Spudich, J. A., Kuczmarski, E. R., Pardee, J. D., Simpson, P. A., Yamamoto, K., Stryer, L. 1981; 46: 553-561

  View details for Web of Science ID A1981NV38800006

• FLOW OF INFORMATION IN THE LIGHT-TRIGGERED CYCLIC-NUCLEOTIDE CASCADE OF VISION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Fung, B. K., Hurley, J. B., Stryer, L. 1981; 78 (1): 152-156

  Abstract

  Photolyzed rhodopsin catalyzes the exchange of GTP for FDP bound to a protein in retinal rod outer segments. We previously proposed that the GTP complex of this protein regulates the cyclic GMP phosphodiesterase and that it may be the first amplified intermediate in visual excitation [Fung, B. K.-K. & Stryer, L. (1980) Proc. Natl. Acad. Sci. USA 77, 2500-2504]. We report here the identification and characterization of transducin, a regulatory protein consisting of three kinds of polypeptide chains: T alpha (39 kilodaltons), T beta (36 kilodaltons), and T gamma (approximately 10 kilodaltons). Reconstituted membranes containing transducin and rhodopsin but no phosphodiesterase exhibit GTPase activity and amplified binding of guanosine 5'[beta, gamma-imido]triphosphate (p[NH]ppG), a nonhydrolyzable analog of GTP, on illumination. A single photolyzed rhodopsin molecule led to the uptake of p[NH]ppG by 71 molecules of transducin. High-pressure liquid chromatography showed that the binding site for GTP is on the alpha subunit of transducin. The isolation of the complex of ;[NH]ppG with T alpha enabled us to determine whether this species is the activator of the phosphodiesterase. We found that phosphodiesterase on unilluminated disc membranes can indeed be fully activated by addition of T alpha containing bound p[NH]ppG. These findings strongly suggest that transducin is the first amplified information-carrying intermediate in the cyclic nucleotide cascade of vision.

  View details for Web of Science ID A1981LA96300025

  View details for PubMedID 6264430

• RETINAL CHROMOPHORE OF RHODOPSIN PHOTOISOMERIZES WITHIN PICOSECONDS SCIENCE Hayward, G., Carlsen, W., SIEGMAN, A., Stryer, L. 1981; 211 (4485): 942-944

  Abstract

  A new picosecond resonance Raman technique shows that resonance Raman lines characteristic of a distorted all-trans retinal appear within 30 picoseconds after photolysis of rhodopsin or isorhodopsin. This finding suggests that isomerization is nearly complete within picoseconds of the absorption of a photon.

  View details for Web of Science ID A1981LC34000027

  View details for PubMedID 7466366

• 1ST STAGE OF AMPLIFICATION IN THE CYCLIC-NUCLEOTIDE CASCADE OF VISION CURRENT TOPICS IN MEMBRANES AND TRANSPORT Stryer, L., Hurley, J. B., Fung, B. K. 1981; 15: 93-108

  View details for Web of Science ID A1981MS71800006

• TRANSDUCIN - AN AMPLIFIER PROTEIN IN VISION TRENDS IN BIOCHEMICAL SCIENCES Stryer, L., Hurley, J. B., Fung, B. K. 1981; 6 (9): 245-247

  View details for Web of Science ID A1981MH85600012

• NEUTRON-DIFFRACTION ANALYSIS OF THE STRUCTURE OF ROD PHOTORECEPTOR-MEMBRANES IN INTACT RETINAS JOURNAL OF MOLECULAR BIOLOGY Yeager, M., SCHOENBORN, B., Engelman, D., Moore, P., Stryer, L. 1980; 137 (3): 315-348

  View details for Web of Science ID A1980JJ50100005

  View details for PubMedID 6973637

• TRANSVERSE LOCATION OF THE RETINAL CHROMOPHORE OF BACTERIORHODOPSIN IN THE PURPLE MEMBRANE THOMAS, D. D., Stryer, L. FEDERATION AMER SOC EXP BIOL. 1980: 1847–47

  View details for Web of Science ID A1980JP62701294

• PHOTOLYZED RHODOPSIN CATALYZES THE EXCHANGE OF GTP FOR BOUND GDP IN RETINAL ROD OUTER SEGMENTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Fung, B. K., Stryer, L. 1980; 77 (5): 2500-2504

  Abstract

  We have studied the binding of guanyl nucleotides to retinal rod outer segment membranes to determine how light activates a cyclic GMP phosphodiesterase and a GTPase. We found that rod outer segment membranes contain tightly bound radioactive GDP after incubation in the dark with [3H]GDP or [alpha-32P]GTP. Reconstituted membranes containing only rhodopsin and phospholipid bind almost no GDP. More than 80% of the radioactive GDP bound to rod outer segment membranes could be released by subsequent illumination. At low light levels, the rate and extent of GDP release were markedly enhanced by the presence of GTP or p[NH]ppG, a nonhydrolyzable analog of GTP. The kinetics of binding of p[NH]ppG paralleled the kinetics of release of bound GDP, indicating that p[NH]ppG was exchanged for bound GDP. The maximal amount of bound p[NH]ppG was 1 per 30 rhodopsins when photolyzed membranes were incubated with 10 micro M nucleotide. Under these conditions, p[NH]ppG binding was half-maximal when only 1 in 90,000 rhodopsins was photolyzed. This corresponds to the catalyzed exchange of 500 p[NH]ppG for bound GDP per photolyzed rhodopsin. We propose a light-activated GTP-GDP amplification cycle involving a guanyl nucleotide binding protein with GTPase activity (E). The essence of this cycle is that photolyzed rhodopsin catalyzes the formation of E . GTP from E . GDP (the major species in the dark) by nucleotide exchange. The formation of several hundred E . GTP per photolyzed rhodopsin may be the first stage of amplification in visual excitation.

  View details for Web of Science ID A1980JU56800026

  View details for PubMedID 6930647

• GRAMICIDIN-A CRYSTALS CONTAIN 2 CATION BINDING-SITES PER CHANNEL NATURE Koeppe, R. E., Berg, J. M., Hodgson, K. O., Stryer, L. 1979; 279 (5715): 723-725

  View details for Web of Science ID A1979GZ95900041

  View details for PubMedID 88018

• SUB-NANOSECOND MOTIONS OF TRYPTOPHAN RESIDUES IN PROTEINS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Munro, I., Pecht, I., Stryer, L. 1979; 76 (1): 56-60

  Abstract

  The dynamics of protein molecules in the subnanosecond and nanosecond time range were investigated by time-resolved fluorescence polarization spectroscopy. Synchrotron radiation from a storage ring was used as a pulsed light source to excite the single tryptophan residue in a series of proteins. The full width at half maximum of the detected light pulse was 0.65 nsec, making it feasible to measure emission anisotropy kinetics in the subnanosecond time range and thereby to resolve internal rotational motions. The proteins investigated exhibit different degrees of rotational freedom of their tryptophan residue, ranging from almost no mobility to nearly complete freedom in the subnanosecond time range. The tryptophan residue of Staphylococcus aureus nuclease B (20,000 daltons) has a single rotational correlation time (varphi) of 9.9 nsec at 20 degrees C, corresponding to a rotation of the whole protein molecule. By contrast, bovine basic A1 myelin protein (18,000 daltons) exhibits varphi of 0.09 and 1.26 nsec, showing that the tryptophan residue in this protein is highly flexible. The single tryptophan of human serum albumin (69,000 daltons) has almost no rotational freedom at 8 degrees C (varphi = 31.4 nsec), whereas at 43 degrees C it rotates rapidly (varphi(1) = 0.14 nsec) within a cone of semiangle 26 degrees in addition to rotating together with the whole protein (varphi(2) = 14 nsec). Of particular interest in the large angular range (semiangle, 34 degrees ) and fast rate (varphi(1) = 0.51 nsec) of the rotational motion of the tryptophan residue in Pseudomonas aeruginosa azurin (14,000 daltons). This residue is known to be located in the hydrophobic interior of the protein. The observed amplitudes and rates of these internal motions of tryptophan residues suggest that elementary steps in functionally significant conformational changes may take place in the subnanosecond time range.

  View details for Web of Science ID A1979GF88500014

  View details for PubMedID 284374

• BIOMEDICAL INSTRUMENTATION DEVELOPMENT - RECOMMENDATIONS OF A WORKSHOP ON THE STATUS AND FUTURE OF BIOPHYSICAL AND BIOCHEMICAL INSTRUMENTATION .1. AMERICAN LABORATORY Abbrecht, P. H., BADMAN, W. S., Cassman, M., Stryer, L. 1979; 11 (9): 82-?

  View details for Web of Science ID A1979HM90500007

• FLUORESCENCE ENERGY-TRANSFER AS A SPECTROSCOPIC RULER ANNUAL REVIEW OF BIOCHEMISTRY Stryer, L. 1978; 47: 819-846

  View details for Web of Science ID A1978FH01600024

  View details for PubMedID 354506

• CONFORMATIONAL CHANGE OF GRAMICIDIN A UPON BINDING OF ALKALI CATIONS - X-RAY CRYSTALLOGRAPHIC AND FLUORESCENCE STUDIES Koeppe, R. E., Hodgson, K. O., Stryer, L. FEDERATION AMER SOC EXP BIOL. 1978: 1726–26

  View details for Web of Science ID A1978EX18302523

• SURFACE DENSITY DETERMINATION IN MEMBRANES BY FLUORESCENCE ENERGY-TRANSFER BIOCHEMISTRY Fung, B. K., Stryer, L. 1978; 17 (24): 5241-5248

  View details for Web of Science ID A1978FX81800025

  View details for PubMedID 728398

• TRANSGLUTAMINASE-CATALYZED INSERTION OF A FLUORESCENT-PROBE INTO PROTEASE-SENSITIVE REGION OF RHODOPSIN BIOCHEMISTRY Pober, J. S., IWANIJ, V., Reich, E., Stryer, L. 1978; 17 (11): 2163-2169

  View details for Web of Science ID A1978FA97500021

  View details for PubMedID 27209

• HELICAL CHANNELS IN CRYSTALS OF GRAMICIDIN-A AND OF A CESIUM-GRAMICIDIN-A COMPLEX - X-RAY-DIFFRACTION STUDY JOURNAL OF MOLECULAR BIOLOGY Koeppe, R. E., Hodgson, K. O., Stryer, L. 1978; 121 (1): 41-54

  View details for Web of Science ID A1978FB39600003

  View details for PubMedID 77905

• FLUORESCENCE ENERGY-TRANSFER IN RAPID-DIFFUSION LIMIT PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA THOMAS, D. D., CARLSEN, W. F., Stryer, L. 1978; 75 (12): 5746-5750

  Abstract

  Energy transfer is enhanced by translational diffusion of the donor and acceptor [Steinberg, I. Z. & Katchalski, E. (1968) J. Chem. Phys. 48, 2404-2410]. The effect of diffusion on energy transfer depends on Dtau(0)/s(2), in which D is the sum of the diffusion coefficients of the donor and acceptor, tau(0) is the lifetime of the donor in the absence of transfer, and s is the mean distance between donors and acceptors. In most previous studies, Dtau(0)/s(2) < 1, corresponding to the static limit. We report here steady-state and kinetic fluorescence experiments showing that Dtau(0)/s(2) > 1, the rapid-diffusion limit, can be attained by using Tb(3+) chelated to dipicolinate as a long-lived energy donor (tau(0) = 2.2 msec). The concentration of rhodamine B, the energy acceptor, resulting in 50% transfer was 0.67 muM, which is three orders of magnitude less than the concentration giving 50% transfer in the static limit. The dependence of the transfer efficiency on diffusion coefficients varying from 5 x 10(-11) to 1.5 x 10(-4) cm(2)/sec, spanning the range from the static limit to the rapid-diffusion limit, is in excellent agreement with theory. It is evident that energy donors with millisecond or longer excited state lifetimes can be used to probe translational motions in membranes and other assemblies. Energy transfer in the rapid diffusion limit is sensitive to the distance of closest approach (a) of the donor and acceptor. For a Tb.(DPA)(3) chelate trapped inside the aqueous space of a membrane vesicle containing eosin phosphatidylethanolamine, a = 10 A. The transverse location of chromophores in model membranes and biological membranes can be determined by this technique.

  View details for PubMedID 16592590

• DIMERIC GRAMICIDIN A TRANSMEMBRANE CHANNEL IN 3 CRYSTAL FORMS Koeppe, R. E., Hodgson, K. O., Stryer, L. ROCKEFELLER UNIV PRESS. 1977: A208–A208

  View details for Web of Science ID A1977DY90100602

• DIMERIC NATURE OF GRAMICIDIN-A TRANSMEMBRANE CHANNEL - CONDUCTANCE AND FLUORESCENCE ENERGY-TRANSFER STUDIES OF HYBRID CHANNELS JOURNAL OF MOLECULAR BIOLOGY VEATCH, W., Stryer, L. 1977; 113 (1): 89-102

  View details for Web of Science ID A1977DM10700005

  View details for PubMedID 69713

• RETINAL HAS A HIGHLY DIPOLAR VERTICALLY EXCITED SINGLET-STATE - IMPLICATIONS FOR VISION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mathies, R., Stryer, L. 1976; 73 (7): 2169-2173

  Abstract

  We have measured the effect of an intense electric field on the absorption spectrum of solutions of all-trans retinal, its unprotonated Schiff base with n-butylamine, and the Cl- salt of this protonated Schiff base. The field-induced change in extinction coefficient as a function of wavelength was analyzed to determine the ground-state dipole moment (mug), the change in dipole moment on excitation (deltamu), and the direction of mug and deltamu). These experiments have shown that all three species become highly dipolar upon excitation to the first allowed excited singlet state (deltamu = 15.6, 9.9, 12D, respectively). The ground-state and excited-state dipole moments are nearly parallel to the long axis of these molecules. Excitation is accompanied by a shift of negative charge toward the carbonyl or Schiff base terminus, making the ionone end of these molecules positively charged. The large excited state dipole moment of all-trans retinal indicates that the vertically excited state, which is of 1Bu parentage (C2h), has become significantly mixed with even-parity states. On the basis of previous theoretical calculations, this mixing is expected to facilitate isomerization in the singlet manifold. We have also found that 11-cis retinal has a large deltamu (12.7 +/- 1.4 D) on excitation. In the visual pigments, the interaction of the excited-state dipole moment of retinal with a suitably located charged group could control the position of the absorption maximum. Also, the large shift in charge density upon excitation of retinal may lead to new electrostatic interactions between the chromophore and the protein that would act as a driving force for the initial conformational changes in visual excitation.

  View details for Web of Science ID A1976BY59800001

  View details for PubMedID 1065867

• RAPID-FLOW RESONANCE RAMAN-SPECTROSCOPY OF PHOTOLABILE MOLECULES - RHODOPSIN AND ISORHODOPSIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mathies, R., Oseroff, A. R., Stryer, L. 1976; 73 (1): 1-5

  Abstract

  We have devised a method for obtaining the resonance Raman spectrum of a photolabile molecule before it is modified by light. The essence of this technique is that the sample is flowed through the light beam at a sufficiently high velocity so that the fraction of photoisomerized (or photodestroyed) molecules in the illuminated volume is very low. This rapid-flow technique has enabled us to measure the resonance Raman spectrum of unphotolyzed bovine rhodopsin in Ammonyx LO detergent solution and in sonicated retinal disc membranes. The major features of these spectra, which are very similar to one another, are the protonated Schiff base line near 1660 cm-1, the ethylenic line at 1545 cm-1, lines due to skeletal modes at 1216, 1240, and 1270 cm-1, and a line due to C-H bending at 971 cm-1. The resonance Raman spectrum of unphotolyzed isorhodopsin formed by the addition of 9-cis-retinal to opsin was also measured. The spectrum of isorhodopsin is more complex and differs markedly from that of rhodopsin. In isorhodopsin, the ethylenic line is shifted to 1550 cm-1, and there are six lines between 1153 and 1318 cm-1. The rapid-flow technique described here makes it feasible to control the extent of interaction between light and any photolabile molecule. We present a theory for predicting the effective sample composition in the illuminated volume as a function of the flow rate, light intensity, and spectral characteristics of the photolabile species.

  View details for Web of Science ID A1976BD04500001

  View details for PubMedID 1061102

• Letter to the editor: Light dissociates enzymatically-cleaved rhodopsin into two different fragments. Journal of molecular biology Pober, J. S., Stryer, L. 1975; 95 (3): 477-481

  View details for PubMedID 1171252

• SIMULTANEOUS FLUORESCENCE AND CONDUCTANCE STUDIES OF PLANAR BILAYER MEMBRANES CONTAINING A HIGHLY ACTIVE AND FLUORESCENT ANALOG OF GRAMICIDIN-A JOURNAL OF MOLECULAR BIOLOGY VEATCH, W. R., Mathies, R., Eisenberg, M., Stryer, L. 1975; 99 (1): 75-92

  View details for Web of Science ID A1975BA17200006

  View details for PubMedID 54431

• FLUORESCENCE SPECTROSCOPY OF AN ORIENTED MODEL MEMBRANE (PROBES/POLARIZATION/OXIDIZED CHOLESTEROL BILAYER/SPHERICAL MEMBRANES) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yguerabide, J., Stryer, L. 1971; 68 (6): 1217-?

  Abstract

  We have devised a simple method that makes it feasible to apply fluorescence techniques to lipid bilayer membranes to elucidate aspects of their structure and dynamics. Fluorescence excitation, emission, and polarization spectra were obtained from a single spherical bilayer membrane consisting of oxidized cholesterol and fluorescent probe. The emission transition moments of N,N'-di(octadecyl)oxacarbocyanine and 12-(9-anthroyl)-stearic acid were found to be aligned parallel to the plane of the bilayer, whereas that of p-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene was aligned in a perpendicular direction. All three probes exhibited appreciable rotational mobility, parallel to the plane of the bilayer, in durations of nanoseconds. An attractive feature of this model membrane is that fluorescence measurements can be made at the same time as electrical measurements and perturbations. Also, it may be possible to incorporate functional protein assemblies into this model and to use fluorescence spectroscopy to delineate some aspects of their assembly and function.

  View details for Web of Science ID A1971J579400033

  View details for PubMedID 5288369

• SEGMENTAL FLEXIBILITY IN AN ANTIBODY MOLECULE JOURNAL OF MOLECULAR BIOLOGY Yguerabide, J., Epstein, H. F., Stryer, L. 1970; 51 (3): 573-?

  View details for Web of Science ID A1970H124400009

  View details for PubMedID 5492607

• DEPENDENCE OF KINETICS OF SINGLET-SINGLET ENERGY TRANSFER ON SPECTRAL OVERLAP PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Haugland, R. P., Yguerabide, J., Stryer, L. 1969; 63 (1): 23-?

  Abstract

  Electronic excitation energy can be transferred between chromophores separated by distances of the order of 30 A. Förster proposed that the transfer occurs by a dipole-dipole resonance interaction which depends on certain spectroscopic and geometric properties of the donor-acceptor pair. His prediction that the rate of transfer depends on the inverse sixth power of the distance between the chromophores was verified previously. In this work, we tested a second prediction of Förster's theory, namely, that the transfer rate is proportional to J, the magnitude of the overlap between the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor.The energy donor was an N-methylindole moiety, and the acceptor was a ketone. These chromophores were fused to a rigid steroid that separated them by 10.2 A. Rate constants for singlet-singlet energy transfer in this system were obtained by nanosecond flash spectroscopy. J was varied over a 40-fold range simply by altering the solvent. We found that the transfer rate is proportional to J, as predicted by Förster's theory. The results bear on the potential use of this energy transfer process to measure distances in biological macromolecules. It is evident that the length of such a spectroscopic ruler can readily be controlled by varying the magnitude of the spectral overlap integral of the energy donor-acceptor pair.

  View details for Web of Science ID A1969D694400004

  View details for PubMedID 16591747

• Energy Transfer: a Spectroscopic Ruler Proc. Natl. Acad. Sci. Stryer, L.,, Haugland, R.P. 1967; 58: 719-725