The Boxer Lab
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Research in the Boxer Lab
My laboratory investigates the structure and function of biological systems using many tools and methods and with a strong physical perspective.  We invent experimental methods and develop theory as needed, and use a wide range of chemical and recombinant DNA methods to modify proteins and monitor or modulate their function.  Several interconnected themes are being pursued and current focus areas are sketched below, linked to further detailed descriptions of individual projects and relevant references [numbers refer to the overall publication list]. 
Energy and Electron Transfer Dynamics in Photosynthetic Reaction Centers
Reaction Center
  We have a long-standing interest in the mechanism of light-driven long-distance electron transfer in photosynthetic reaction centers, one of the fastest known reactions.  This is being studied by femtosecond fluorescence and transient absorption spectroscopy, manipulation in electric fields, site-specific and global mutagenesis and some novel types of Stark spectroscopy we have developed and applied to many types of molecules.  Current work focuses on novel bacterial reaction centers that lack normal electron acceptors so that alternate pathways of electron transfer can be probed in depth [236, 264].  Photosynthesis is responsible for all biological energy storage in the biosphere and offers the ultimate model system for solar energy conversion in non-biological systems.
Excited State Dynamics in Green Fluorescent Protein (GFP)
  GFP is widely used as a probe for the location of proteins in cells and as a sensor, e.g. for pH, metal ions and proteases.  Our lab was the first to demonstrate that the GFP chromophore exists in two protonation states and that these states can be interconverted by ultrafast excited state proton transfer [136].  This concept has since been developed in many labs, including ours, to generate novel GFP variants with diverse colors and sensitivities to their environment.  Current work focuses on split GFP in which individual structural elements such as entire beta strands or the central chromophore-containing helix are replaced with synthetic elements [259].  In this way non-natural amino acids can be introduced and used to characterize the excited states of the chromophore, to introduce novel functionality, and to probe beta barrel assembly and chromophore formation mechanisms.
Electrostatics and Dynamics in Proteins
  Beginning with our work on photosynthetic reaction centers, we are broadly interested in electrostatics in proteins and how electrostatics affects function.  Early test systems involved mutants of myoglobin, which was first cloned and expressed in our laboratory [34, 57].  This led to the concept of probes whose sensitivity to electric fields can be calibrated by Stark spectroscopy — spectroscopy in electric fields — which we have developed into a broadly applicable spectroscopic method [262].  Vibrational Stark experiments are particularly useful as molecular vibrations can be exploited as local and directional probes for mapping electrostatic fields in proteins [219].  Current work focuses on the application of nitrile probes introduced into proteins on inhibitors, by introduction of non-natural amino acids or by modification of amino acids, e.g. the conversion of cysteine –SH to thiocyanate –SCN [243].  Thiocyanate probes can be introduced at any site in a protein by this simple chemical modification, and this has been used to probe the active site of the enzyme ketosteroid isomerase (KSI) in collaboration with the Herschlag lab [247].  IR spectral shifts for these probes and probes on inhibitors in the enzyme human aldose reductase, when mutants are made or protein conformational changes occur, are compared quantitatively with high-level simulations as fundamental tests of the validity of widely used electrostatics calculations [239].  These probes are also ideally suited for vibrational coherence measurements, and this is being pursued in collaboration with the Fayer lab.  Probes have also been developed that can measure the time-dependent solvation of charges at different positions in proteins [208], a key aspect of protein-protein and protein-ligand interactions and catalysis.
Model Membranes    
  Our group has developed supported lipid bilayers as mimics for cell surfaces and as tools in biotechnology.  A broad vision is to engineer interfaces between hard surfaces and soft materials, ultimately leading to sophisticated biocompatible interfaces that can be used to control, interrogate or organize complex living systems.  We have developed methods for partitioning and manipulating the composition and organization of these unique self-assembled systems [142, 170, 206], and these methods are now used in many laboratories. 
Recent work addresses four interrelated areas: (1) characterization of membrane organization and domains and protein association with these domains using a novel type of imaging mass spectrometry [244]; (2) models for membrane fusion using DNA-tethered vesicles and DNA-lipid conjugates as surrogates for the protein machinery that controls membrane fusion in vivo [261]; (3) the development of novel types of tethered lipid bilayers that can be used as a platform for studying membrane domains, membrane topology in model membrane junctions, and vesicle fusion [266]; and (4) a membrane interferometer in which a free-standing lipid bilayer is held within a few hundred nm of an atomically flat mirror [265] with the ultimate goal of measuring protein conformational changes optically with sub-nm precision in parallel with electrical measurements, e.g. in ion channels.
The Boxer LaboratoryStanford UniversityDepartment of Chemistry • 380 Roth Way, Stanford, California, 94305-5012 • (650) 723-4482
Questions about this website may be directed to Debra Frank.