zes the activities currently supported by two NIGMS grants and my vision for the evolution of this research over the next five years. The common theme that unifies this research is a unique combination of the development and application of new physical methods that can be broadly applied to the quantitative analysis of biological systems. The long-term goals of GM27738 (Electrostatics and Dynamics in Proteins) are to develop spectroscopic methods for probing electric fields in proteins and to apply these methods to obtain quantitative information on fields and their effects on function at the active sites of several enzymes. We have led the development of vibrational Stark spectroscopy as a general approach to map these fields. Recent and continuing work emphasizes the connection between electrostatics and catalysis where we can, for the first time, estimate the electrostatic contribution to the catalytic proficiecy of enzymes. We also study dynamics in green fluorescent protein (GFP), which we began to study reasoning that the native chromophore could be used to probe solvation dynamics (time dependent electric fields) in proteins. Instead we discovered that GFP exhibits excited state proton transfer. Recent work emphasizes novel applications of split GFP where light-driven association and dissociation reactions have been discovered. We seek to understand the basic mechanism(s) of these processes and optimize them for optogenetic and imaging applications. The long-term goals of GM069630 (Membrane Fusion and Dynamics Using Supported Bilayers) are to develop methods to probe the organization and dynamic reorganization of lipids and proteins in biological membranes and to apply these methods to problems of broad biological importance. Our lab has pioneered the development of model membrane architectures, along with imaging and analytical methods, that probe fundamental aspects of membrane organization and dynamics. We use these approaches to address open questions in three areas of current biological significance. (i) The mechanism of vesicle and enveloped viral membrane fusion is studied using model membrane architectures we developed as targets to precisely probe the steps of fusion at the single event level. (ii) The organization of lipids and membrane proteins are characterized with unprecedented lateral resolution using imaging mass spectrometry, emphasizing the correlated motion of lipid and protein components believed to be associated in rafts. (iii) A novel membrane interferometer has been designed with the ultimate goal of correlated measurements of integral membrane protein conformational changes by optical interferometry and electrical activity by electrophysiological methods. Each of these areas represents a major current challenge in membrane biophysics and biology.

Public Health Relevance

We are developing methods that probe the electrostatic field at the active site of enzymes, often using drugs that display the probes that sense the field. This is a new quantitative approach for discriminating active sites in targets of direct biomedical relevance and creating enzymes with new functions. New types of green fluorescent protein (GFP) variants are been developed that can be used both for imaging and to modulate cell physiology. We also develop new methods for studying membranes and membrane-associated proteins that can impact our understanding of biological function and organization, as well as impact biotechnology.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1)
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Flicker, Paula F
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Stanford University
Schools of Arts and Sciences
United States
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Moss 3rd, Frank R; Shuken, Steven R; Mercer, Jaron A M et al. (2018) Ladderane phospholipids form a densely packed membrane with normal hydrazine and anomalously low proton/hydroxide permeability. Proc Natl Acad Sci U S A 115:9098-9103
Goronzy, I N; Rawle, R J; Boxer, S G et al. (2018) Cholesterol enhances influenza binding avidity by controlling nanoscale receptor clustering. Chem Sci 9:2340-2347
Rawle, Robert J; Webster, Elizabeth R; Jelen, Marta et al. (2018) pH Dependence of Zika Membrane Fusion Kinetics Reveals an Off-Pathway State. ACS Cent Sci 4:1503-1510
Deng, Alan; Boxer, Steven G (2018) Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag. J Am Chem Soc 140:375-381
Schneider, Samuel H; Kratochvil, Huong T; Zanni, Martin T et al. (2017) Solvent-Independent Anharmonicity for Carbonyl Oscillators. J Phys Chem B 121:2331-2338
Boxer, Steven G (2017) Comment on ""Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes"". J Phys Chem B 121:7395-7396
Fried, Stephen D; Boxer, Steven G (2017) Electric Fields and Enzyme Catalysis. Annu Rev Biochem 86:387-415
Lin, Chi-Yun; Both, Johan; Do, Keunbong et al. (2017) Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs). Proc Natl Acad Sci U S A 114:E2146-E2155
Schneider, Samuel H; Boxer, Steven G (2016) Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site. J Phys Chem B 120:9672-84
Moss 3rd, Frank R; Boxer, Steven G (2016) Atomic Recombination in Dynamic Secondary Ion Mass Spectrometry Probes Distance in Lipid Assemblies: A Nanometer Chemical Ruler. J Am Chem Soc 138:16737-16744

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