My laboratory investigates the structure and function of biological systems with a strong physical perspective. We invent experimental methods and develop theory as needed. We are pursuing several interconnected themes:
Excited State Dynamics in GFP & Split GFP
Green Fluorescent Protein (GFP) is widely used as a probe to localize proteins in cells. Our lab was the first to demonstrate that the GFP chromophore exists in two protonation states, interconvertible by ultrafast excited state proton transfer. We and others have since developed this idea to generate novel GFP variants with diverse colors and sensitivities. Current work focuses on split GFP in which structural elements such as entire beta strands are replaced with synthetic ones. We have discovered that some split GFPs can be photodissociated, generating a peptide and a truncated protein; alternatively, in some conditions, light can be used to associate peptides with truncated proteins. Thus these split GFPs are optogenetic elements for manipulations inside cells, and we seek to understand how they work.
Electrostatics and Dynamics in Proteins
We study electrostatics in proteins and how electric fields affect function. Early test systems used mutants of myoglobin, which was first cloned and expressed in our lab. This led to probes whose sensitivity to electric fields can be calibrated by Stark spectroscopy — spectroscopy in electric fields — which we have developed into a broadly applicable method. Vibrational Stark experiments exploit molecular vibrations as local and directional probes to map electrostatic fields in proteins. Current work applies nitrile probes introduced into proteins on inhibitors. These can be used to probe electrostatics and hydration at the active sites of important drug targets. Recent work focuses on carbonyl probes to study enzymatic reactions. By combining the vibrational Stark effect, vibrational solvatochromism and MD simulations, we have developed a general method to measure the absolute field sensed by the carbonyl probe in proteins. This has been used to quantify the electrostatic contribution to the catalytic rate in several enzymes.
Our group has developed supported lipid bilayers as mimics for cell surfaces and 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 to partition and manipulate elements of these unique self-assembled systems; these methods are now used in many laboratories.
Recent work addresses four interrelated areas: 1) characterization of membrane organization, domains and protein associations using a novel type of imaging mass spectrometry; 2) models for membrane fusion and investigations into the fusion of enveloped viruses to their target membrane; 3) development of tethered lipid bilayers as a platform to study membrane domains, junction topology, vesicle fusion and enveloped virus fusion; and 4) a membrane interferometer where a free-standing lipid bilayer is held within a few hundred nm of an atomically flat mirror, with the ultimate goal of measuring protein conformational changes optically with sub-nm precision in parallel with electrical measurements, e.g. in ion channels.
Energy and Electron Transfer in Photosynthesis
Light-driven long-distance electron transfer in photosynthetic reaction centers is one of the fastest known chemical reactions. We study this by femtosecond fluorescence and transient absorption spectroscopy, manipulation in electric fields, site-specific and global mutagenesis and some novel types of Stark spectroscopy. Current work probes alternate pathways of electron transfer in novel bacterial reaction centers that lack normal electron acceptors, and introduces non-canonical amino acids to perturb and probe pathways.
Levinson, N. M., & Boxer, S. G. (2014). A conserved water-mediated hydrogen bond network defines bosutinib's kinase selectivity. NATURE CHEMICAL BIOLOGY, 10(2), 127-132.
Wu, Y., & Boxer, S. G. (2016). A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase. Journal of the American Chemical Society, 138(36), 11890–95.
Hughes, L. D., Rawle, R. J., & Boxer, S. G. (2014). Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers. PloS one, 9(2).
Lozano, M. M., Liu, Z., Sunnick, E., Janshoff, A., Kumar, K., & Boxer, S. G. (2013). Colocalization of the Ganglioside G(M1) and Cholesterol Detected by Secondary Ion Mass Spectrometry. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 135(15), 5620–30.
Rawle, R. J., Boxer, S. G., & Kasson, P. M. (2016). Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates. Biophysical journal, 111(1), 123-131.
Lozano, M. M., Hovis, J. S., Moss, F. R., & Boxer, S. G. (2016). Dynamic Reorganization and Correlation among Lipid Raft Components. Journal of the American Chemical Society, 138(31), 9996-10001.
Fried, S. D., Bagchi, S., & Boxer, S. G. (2014). Extreme electric fields power catalysis in the active site of ketosteroid isomerase. SCIENCE, 346(6216), 1510-1514.
Do, K., & Boxer, S. G. (2013). GFP Variants with Alternative beta-Strands and Their Application as Light-driven Protease Sensors: A Tale of Two Tails. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 135(28), 10226-10229.
van Lengerich, B., Rawle, R. J., Bendix, P. M., & Boxer, S. G. (2013). Individual Vesicle Fusion Events Mediated by Lipid-Anchored DNA. BIOPHYSICAL JOURNAL, 105(2), 409-419.
Fried, S. D., & Boxer, S. G. (2015). Measuring Electric Fields and Noncovalent Interactions Using the Vibrational Stark Effect. ACCOUNTS OF CHEMICAL RESEARCH, 48(4), 998-1006.