Steven Boxer, Doctoral Dissertation Advisor (AC)
Electrostatic control of photoisomerization pathways in proteins.
Science (New York, N.Y.)
2020; 367 (6473): 76–79
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
Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins
Journal of the American Chemical Society
2019; 141 (38): 15250-15265
View details for DOI 10.1021/jacs.9b07152
Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs).
Proceedings of the National Academy of Sciences of the United States of America
Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.
View details for DOI 10.1073/pnas.1618087114
View details for PubMedID 28242710
View details for PubMedCentralID PMC5358378
Isolation and Identification of Post-Transcriptional Gene Silencing-Related Micro-RNAs by Functionalized Silicon Nanowire Field-effect Transistor
Many transcribed RNAs are non-coding RNAs, including microRNAs (miRNAs), which bind to complementary sequences on messenger RNAs to regulate the translation efficacy. Therefore, identifying the miRNAs expressed in cells/organisms aids in understanding genetic control in cells/organisms. In this report, we determined the binding of oligonucleotides to a receptor-modified silicon nanowire field-effect transistor (SiNW-FET) by monitoring the changes in conductance of the SiNW-FET. We first modified a SiNW-FET with a DNA probe to directly and selectively detect the complementary miRNA in cell lysates. This SiNW-FET device has 7-fold higher sensitivity than reverse transcription-quantitative polymerase chain reaction in detecting the corresponding miRNA. Next, we anchored viral p19 proteins, which bind the double-strand small RNAs (ds-sRNAs), on the SiNW-FET. By perfusing the device with synthesized ds-sRNAs of different pairing statuses, the dissociation constants revealed that the nucleotides at the 3'-overhangs and pairings at the terminus are important for the interactions. After perfusing the total RNA mixture extracted from Nicotiana benthamiana across the device, this device could enrich the ds-sRNAs for sequence analysis. Finally, this bionanoelectronic SiNW-FET, which is able to isolate and identify the interacting protein-RNA, adds an additional tool in genomic technology for the future study of direct biomolecular interactions.
View details for DOI 10.1038/srep17375
View details for Web of Science ID 000365425900002
View details for PubMedID 26616332
View details for PubMedCentralID PMC4663627