All Publications


  • Structural and spectroscopic characterization of photoactive yellow protein and photoswitchable fluorescent protein constructs containing heavy atoms. Journal of photochemistry and photobiology. A, Chemistry Romei, M. G., Lin, C., Boxer, S. G. 2020; 401

    Abstract

    Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore's photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-theart time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization.

    View details for DOI 10.1016/j.jphotochem.2020.112738

    View details for PubMedID 32753830

  • Electrostatic control of photoisomerization pathways in proteins. Science (New York, N.Y.) Romei, M. G., Lin, C. Y., Mathews, I. I., Boxer, S. G. 2020; 367 (6473): 76–79

    Abstract

    Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design.

    View details for DOI 10.1126/science.aax1898

    View details for PubMedID 31896714

  • Split Green Fluorescent Proteins: Scope, Limitations, and Outlook ANNUAL REVIEW OF BIOPHYSICS, VOL 48 Romei, M. G., Boxer, S. G., Dill, K. A. 2019; 48: 19–44
  • A unified model for photophysical and electro-optical properties of Green Fluorescent Proteins. Journal of the American Chemical Society Lin, C. Y., Romei, M. G., Oltrogge, L. M., Mathews, I. I., Boxer, S. G. 2019

    Abstract

    Green fluorescent proteins (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.

    View details for DOI 10.1021/jacs.9b07152

    View details for PubMedID 31450887

  • Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins. Journal of the American Chemical Society Chang, J., Romei, M. G., Boxer, S. G. 2019

    Abstract

    Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas, in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

    View details for DOI 10.1021/jacs.9b08356

    View details for PubMedID 31533429

  • Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins Journal of the American Chemical Society Lin, C., Romei, M. G., Oltrogge, L. M., Mathews, I. I., Boxer, S. G. 2019; 141 (38): 15250-15265

    View details for DOI 10.1021/jacs.9b07152

  • Photoactive Split Green Fluorescent Protein: Engineering a New Optogenetic and Imaging System Romei, M. G., Longwell, C. K., Cochran, J. R., Boxer, S. G. CELL PRESS. 2018: 177A–178A