Structural biology of transmembrane transport.
The research in my laboratory focuses on understanding the structural and thermodynamic mechanisms by which proteins perform mechanical activities on a molecular scale, with a long-term goal of developing protein machines with novel activities. Biochemical mechanisms designed to achieve mechanical work rely on the interconversion of meta-stable protein structures in a process that is gated by protein-protein or protein-ligand interactions and coupled to some source of free energy such as high-energy phosphodiester bonds or a concentration gradient. Understanding these mechanisms in detail requires knowledge of the three-dimensional structures of the protein domains and the protein-protein and protein-ligand interfaces, as well knowledge of the thermodynamic stablility of the various conformational states along the reaction pathway and the kinetics with which these states are interconverted. Therefore, research in this area lies at the intersection of the fields of protein structure, molecular recognition, and protein dynamics. The tools that are employed in these studies include high-resolution x-ray crystallography to establish static structures and various forms of protein spectroscopy to characterize the kinetics and thermodynamics of complex formation as well as conformational reaction dynamics.
A major emphasis in the lab is on elucidation of the mechanism of protein-mediated transmembrane transport phenomena. As part of my post-doctoral research, I determined the crystal structure of the soluble form of the SecA translocation ATPase, an enzyme that mediates the ATP-driven extrusion of secreted polypeptides through the bacterial plasma membrane (J.F. Hunt, S. Weinkauf, L. Henry, D.B. Oliver, and J. Deisenhofer, manuscript in preparation). This enzyme inserts itself into and through membranes in the course of its ATPase cycle, and it is believed to function as a "molecular ratchet", pulling a piece of protein through the membrane concomittant with its membrane-insertion/retraction cycle. The crystal structure of SecA has led to a model for the intial stages of the transport reaction which will be tested in subsequent experiments. These experiments will include crystal stucture determinations of sub- domains of SecA in complex with other proteins domains required in the transport pathway plus fluorscence studies of SecA-ligand interactions, SecA-membrane interactions, and domain movements within SecA.
Other projects include an effort to develop an aritificial facilitated diffusion machine based on a peptide that spontaneously penetrates phospholipid bilayers and strucural studies of complexes of DnaJ class molecular chaperones.
- Benach J, Lee I, Edstrom W, Kuzin AP, Chiang Y, Acton TB, Montelione GT, Hunt JF. (2003) The 2.3-A Crystal Structure of the Shikimate 5-Dehydrogenase Orthologue YdiB from Escherichia coli Suggests a Novel Catalytic Environment for an NAD-dependent Dehydrogenase J Biol Chem. 278(21): 19176-82.
- Karpowich NK, Huang HH, Smith PC, Hunt JF. (2003) Crystal structures of the BtuF periplasmic-binding protein for vitamin B12 suggest a functionally important reduction in protein mobility upon ligand binding J Biol Chem. 278(10): 8429-34.
- J. F. Hunt, Sevil Weinkauf, Lisa Henry, John J. Fak, Paul McNicholas, Donald B. Oliver, and Johann Deisenhofer (2002) Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA Science 297: 2018-2026. Article
- Keller JP, Smith PM, Benach J, Christendat D, deTitta GT, Hunt JF. (2002) The crystal structure of MT0146/CbiT suggests that the putative precorrin-8w decarboxylase is a methyltransferase Structure 10(11): 1475-87.
- Karpowich, O. Martsinkevish, L. Millen, L., Y.-R. Yuan, .K. MacVey, P.J. Thomas, and J.F. Hunt (2001) Crystal structures of MJ1267 reveal an induced-fit effect at the ATPase active site of an ABC transporter Structure 9: 571-586.
- Y.-R. Yuan, S. Blecker, O. Martsinkevish, L. Millen, P.J. Thomas, and J.F. Hunt (2001) Crystal structure of the MJ0796 ATP-binding cassette: implications for the structural consequences of ATP-hydrolysis in the active site of an ABC transporter J. Biol. Chem. 276: 32313-32321.
- P.J. Thomas and J.F. Hunt (2001) A snapshot of Nature's favorite pump Nature Structural Biology 8: 920-923.
- W.C. Wigley, R.D. Stdiham, N.M. Smith, J.F. Hunt, and P.J Thomas (2001) Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein Nature Biotechnology 19: 131-136.
- J.F. Hunt, S.M. van der Vies, L. Henry, and J. Deisenhofer (1997) Structural adaptations in the specialized bacteriophage T4 co-chaperonin Gp31 expand the size of the Anfinsen cage Cell 90: 361-371.
- J.F. Hunt, F.M.D. Vellieux, and J. Deisenhofer (1997) A connected set algorithm for the identification of spatially contiguous regions in crystallographic envelopes Acta Cryst. D53: 434-437.
- J.F. Hunt, T.N. Earnest, O. Bousche, K. Kalghatgi, K. Reilly, C. Horvath, K.J. Rothschild, and D.M. Engelman (1997) A biophysical study of integral membrane protein folding Biochemistry 36: 15156-15176.
- J.F. Hunt, P. Rath, K.J. Rothschild, and D.M. Engelman (1997) Spontaneous, pH-dependent membrane-insertion of a transbilayer a-helix Biochemistry 36: 15177-15192.
- J.F. Hunt, A.J. Weaver, S.J. Landry, L. Gierasch, and J. Deisenhofer (1996) The crystal structure of the GroES co-chaperonin at 2.8 angstroms resolution Nature 379: 37-45.