研究领域

Biochemistry/Organic Chemistry/Physical Chemistry

Our research is focused on elucidating the structure and function of proteins, with particular emphasis on fluorescent proteins and remodeling enzymes. The ultimate goal is to introduce novel biochemical, optical, or functional features into a protein's scaffold. We utilize macromolecular X-ray crystallography to determine atomic-resolution structures, steady-state spectroscopy to investigate protein maturation kinetics, ultra-fast spectroscopy to examine the photochemistry of intrinsic chromophores, and molecular biology for protein engineering purposes. Protein self-processing reactions: In fluorescent proteins such as GFP, the biosynthesis of several chemically distinct chromophores involves self-catalysis of peptide cyclization and oxidation reactions. Our research aims to develop step-by-step mechanistic models for the amino acid derivatizations that occur spontaneously upon protein folding, yielding brightly colored fluorophores from a single gene product. Structural basis of color evolution in fluorescent proteins: Four color classes (cyan, green, yellow, red) have evolved repeatedly and independently along different lineages of GFP-like proteins. We aim to understand how conformational remodeling facilitates phenotypic changes such as color switching events during protein evolution. Bioenergy and photosynthesis: The regulation of carbon fixation in higher plants and green algae includes remodeling enzymes responsible for maintaining the activity of RuBisCo, which catalyzes the central carboxylation reaction. We are interested in determining the mechanism of action of these auxiliary factors, as they appear to limit the net rate of CO2 fixation at elevated temperatures. Biomedical: We are investigating the structural and biochemical properties of M. tuberculosis protein factors with proposed roles in the regulation of MTB virulence.

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Wachter, R. M., Salvucci, M. E., Carmo-Silva, A. E., Barta, C., Genkhov, T., Spreitzer, R. J. (2013), Activation of Interspecies-hybrid Rubisco Enzymes to Assess Different Models for the Rubisco – Rubisco Activase Interaction Photosynth. Res. (in press) DOI: 10.1007/s11120-013-9827-0. Kennis, J. T. M., van Stokkum, I. H. M., Peterson, D. S., Pandit, A., Wachter, R. M. (2013), Ultrafast Proton Shuttling in Psammocora Cyan Fluorescent Protein. The Journal of Physical Chemistry B (in press) DOI: 10.1021/jp401114e. Watkins, J. L., Kim, H., Markwardt, M. L., Chen, L., Fromme, R., Rizzo, M. A., Wachter, R. M. (2013), The 1.6 Å structure of a FRET-optimized Cerulean Fluorescent Protein. Acta Crystallogr. D69, 767-773. Henderson, J. N., Hazra, S., Dunkle, A. M., Salvucci, M. E., Wachter, R. M. (2013) Biophysical Characterization of Higher Plant Rubisco Activase. Biophys. Biochim. Acta 1834, 87-97. Chakraborty, M., Kuriata, A. M., Henderson, J. N., Salvucci, M. E., Wachter, R. M., Levitus*, M. (2012) Protein Oligomerization Monitored by Fluorescence Fluctuation Spectroscopy: Self-Assembly of Rubisco Activase. Biophys. J. 103, 949-958. Henderson, J. N., Kuriata, A. M., Fromme, R., Salvucci, M. E., Wachter, R. M. (2011) Atomic Resolution X-ray Structure of the Substrate Recognition Domain of Higher Plant Rubisco Activase. J. Biol. Chem. 286, 35683-35688. Markwardt, M. L., Kremers, G.-J., Kraft, C. A., Ray, K., Cranfill, P. J. C., Wilson, K. A., Day, R. N., Wachter, R. M., Davidson, M. W., Rizzo, M. A. (2011) An Improved Cerulean Flourescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching. PlosOne 6, e17896. Wachter, R. M., Watkins, J. L., Kim, H. (2010) Mechanistic Diversity of Red Fluorescence Acquisition by GFP-like Proteins. Current Topics Invited Review. Biochemistry 49, 7417-7427. Barta, C., Dunkle, A. M., Wachter, R. M., Salvucci, M. E. (2010) Structural Changes Associated with the Acute Thermal Instability of Rubisco Activase. Archives of Biochemistry and Biophysics 499, 17-25. Pouwels, L. J., Zhang, L., Chan, N. H., Dorrestein, P. C., Wachter, R. M. (2008) Kinetic Isotope Effect Studies on the de novo Rate of Chromophore Formation in Fast- and Slow-maturing GFP Variants. Biochemistry 47, 10111-10122. Malo, G. D., Wang, M., Wu, D., Stellin, A., Tonge, P. J., Wachter, R. M. (2008) Crystal Structure and Raman Studies of dsFP483, a Cyan Fluorescent Protein from Discosoma striata. J. Mol. Biol. 378, 869-884. Malo, G. D., Pouwels, L. J., Wang, M., Weichsel, A., Montfort, W. R., Rizzo, M. A., Piston, D. W., Wachter, R. M. (2007). X-ray Structure of Cerulean GFP: A Tryptophan-Based Chromophore Useful for Fluorescence Lifetime Imaging. Biochemistry 46, 9865-9873. Accelerated Publication. Wachter, R. M. (2007). Chromogenic Cross-link Formation in Green Fluorescent Protein. Acc. Chem. Res. 40, 120-127 Zhang, L., Patel, H. N., Lappe, J. W., Wachter, R. M. (2006). Reaction Progress of Chromophore Biogenesis in Green Fluorescent Protein. J. Am. Chem. Soc. 128, 4766-4772. Wachter, R. M. (2006). The family of GFP-like proteins: Structure, Function, Photophysics and Biosensor Applications. Introduction and Perspective, Symposium-in-print, Photochem. Photobiol. 82, 339-344 Wachter, R. M. (2006). Mechanistic Aspects of GFP Chromophore Biogenesis. Progress in Biomedical Optics and Imaging 7. Proc. of SPIE Vol. 6098, 609803-1 – 609803-8. Keynote Paper. Wang, M., Patel, H. N., Wachter, R. M. (2005). X-ray Diffraction Analysis and Molecular Replacement Solution of the Cyan Fluorescent Protein dsFP483. Acta Crystallogr. F61, 922-924. Sniegowski, J. A., Phail, M. E., Wachter, R. M. (2005). Maturation Efficiency, Trypsin Sensitivity and Optical Properties of Arg96, Glu222 and Gly67 Variants of Green Fluorescent Protein. Biochem. Biophys. Res. Comm. 332, 657-663. Scruggs, A. W., Flores, C. L., Wachter, R. M., Woodbury, N. W. (2005). Development and Characterization of Green Fluorescent Protein Mutants with Altered Lifetimes. Biochemistry 44, 13377- 13384. Rosenow, M. A., Patel, H. N, Wachter, R. M. (2005). Oxidative Chemistry in the GFP Active Site Leads to Covalent Cross-Linking of a Modified Leucine Side Chain with a Histidine Imidazole: Implications for the Mechanism of Chromophore Formation. Biochemistry 44, 8303-8311. Sniegowski, J. A., Lappe, J. W., Patel, H. N., Huffman, H. A., Wachter, R. M. (2005). Base-catalysis of Chromophore Fromation in Arg96 and Glu222 variants of Green Fluorescent Protein. J. Biol. Chem. 280, 26248-26255. Remington, S. J., Wachter, R. M., Yarbrough, D. K., Branchaud, B. P., Anderson, D. C., Kallio, K., Lukyanov, K. A. (2005). zFP538, a Yellow Fluorescent Protein from Zoanthus, Contains a Novel Three-Ring Chromophore. Biochemistry 44, 202-212. Rosenow, M. A., Huffman, H. A., Phail, M. E., Wachter, R. M. (2004). The Crystal Structure of the Y66L Variant of Green Fluorescent Protein Supports a Cyclization-Oxidation-Dehydration Mechanism for Chromophore Maturation. Biochemistry 43, 4464-4472. Wachter, R. M. (2004). Focus Note: The Art of Disentangling Difference Electron Density Maps: Snapshots of Early States in the Photoactive Yellow Protein Photocycle? Photochem. Photobiol. 80, No.1, ix-x Bell, A. F., Stoner-Ma, D., Wachter, R. M., Tonge, P. J. (2003). Light Driven Decarboxylation of Wild-Type Green Fluorescent Protein. J. Am. Chem. Soc. 125, 6919-6926. Yarbrough, D., Wachter, R. M., Kallio, K., Matz, M. V., Remington, S. J. (2001). Refined Crystal Structure of DsRed, a Red Fluorescent Protein from Coral, at 2.0 Å Resolution. Proc. Natl. Acad. Sci. USA 98, 462-467. Wachter, R. M., Yarbrough, D., Kallio, K., Remington, S. J. (2000). Crystallographic and Energetic Analysis of Binding of Selected Anions to the Yellow Variants of Green Fluorescent Protein. J. Mol. Biol. 301, 159-173. Bell, A. F., He, X., Wachter, R. M., Tonge, P. J. (2000). Probing the Ground State Structure of the Green Fluorescent Protein Chromophore Using Raman Spectroscopy. Biochemistry 39, 4423-4431. Jayaraman, S., Haggie, P., Wachter, R. M., Remington, S. J., Verkman, A. S. (2000). Mechanism and Cellular Applications of a Green Fluorescent Protein-based Halide Sensor. J. Biol. Chem. 275, 6047-6050. Wachter, R. M., Remington, S. J. (1999). Sensitivity of the YFP Form of Green Fluorescent Protein to Halides and Nitrate. Curr. Biol. 9, R628-R629. Elsliger, M.-A., Wachter, R. M., Kallio, K., Hanson, G. T., Remington, S. J. (1999). Structural and Spectral Response of Green Fluorescent Protein Variants to Changes in pH. Biochemistry 38, 5296-5301. Wachter, R. M., Elsliger, M.-A., Kallio, K., Hanson, G. T., Remington, S. J. (1998). Structural Basis of Spectral Shifts in the Yellow-emission Variants (YFPs) of Green Fluorescent Protein. Structure 6, 1267-1277. Wachter, R. M., Branchaud, B. P. (1998). Construction and Analysis of a Semi-quantitative Energy Profile for the Reaction Catalyzed by the Radical Enzyme Galactose Oxidase. Biochim. Biophys. Acta 1384, 43-54. Wachter, R. M., King, B. A., Heim, R., Kallio, K., Tsien, R. Y., Boxer, S. G., Remington, S. J. (1997). Crystal Structure and Photodynamic Behavior of the Blue-emission Variant Y66H/Y145F of Green Fluorescent Protein. Biochemistry 36, 9759-9765. Wachter, R. M., Montague-Smith, M. P., Branchaud, B. P. (1997). Beta-Haloethanol Substrates as Probes for Radical Mechanisms for Galactose Oxidase. J. Am. Chem. Soc. 119, 7743-7749. Wachter, R. M., Branchaud, B. P. (1996). Thiols as Mechanistic Probes for Catalysis by the Free Radical Enzyme Galactose Oxidase. Biochemistry 35, 14425-14435. Wachter, R. M., Branchaud, B. P (1996). Molecular Modeling Studies on Oxidation of Hexopyranoses by Galactose Oxidase. An Active Site Topology Apparently Designed to Catalyze Radical Reactions, Either Concerted or Stepwise. J. Am. Chem. Soc. 118, 2782-2789. Montague-Smith, M. P., Wachter, R. M., Branchaud, B. P. (1992). Preparation of Fully Oxidized Active and Reduced Inactive Forms of Galactose Oxidase from Dactylium dendroides Using Ferricyanide-containing Oxidizing and Ferrocyanide-containing Reducing Forms of Ion Exchange Resins. Anal. Biochem. 207, 353-355.