Within the past two decades, Nuclear Magnetic Resonance spectroscopy (NMR) has become a powerful technique for the study of macromolecular structure and dynamics in the solution state. Research in my lab will be concerned with both the development and application of novel NMR techniques for studying the complex interactions that underlie biological function. Of particular interest are studies of molecular dynamics, the nature of intermolecular recognition and the quaternary organization of multi-domain protein systems.
The full repertoire of multidimensional NMR methodology will be employed to study these problems, including spin relaxation, scalar coupling, NOE, and hydrogen exchange experiments. However, the primary approach will be centered around recently developed techniques for the measurement of Residual Dipolar Couplings (RDCs) in macromolecules. These RDCs, which are normally averaged to zero in solution, are made observable by introducing a very weak degree of alignment of the biomolecule relative to the magnetic field. This alignment is typically achieved by dissolving the protein or nucleic acid along with a suitable co-solute, such as bacteriophage particles. The resulting RDCs are relatively easily measured and represent an abundant source of highly precise information on the relative orientations of different internuclear 'bonds' within the molecule. Intriguingly, RDCs also exhibit sensitivity to molecular motions on the nsec-sec timescales, during which many functionally important motions occur. These motional timescales have traditionally been very difficult to access experimentally, and thus a major objective will be to develop RDC-based techniques to enable the study of these motions.
One of the applications of these techniques will be to investigate the quaternary organization of tetrameric ubiquitin. Tetramers of the protein ubiquitin can assume a multi-faceted role in cellular signal-transduction mechanisms, which depends on how they are linked together. A major goal will be to gain insights into the nature of this important and versatile signal through studies of its solution state conformations. In addition, efforts are ongoing to develop methodology for the simultaneous determination of both the 3-dimensional structure and a detailed description of the dynamics of a protein. A closely related objective is to develop the capability of using NMR to rapidly determine the backbone fold of a protein to moderate resolution, which would represent an important contribution to current structural genomics initiatives.
Research in my lab is concerned with the development and application of novel NMR techniques to understand protein structure and function. In particular we are interested in how molecular dynamics, intermolecular recognition and the quaternary organization of multi-domain proteins serve to mediate biological function. NMR spectroscopy is particularly well suited to study such problems and its capabilities are still evolving. One area of continued research is centered on making NMR measurements when the protein is slightly aligned relative to the magnetic field. Most prominent among the benefits of this approach is the appearance of residual dipolar couplings (RDCs) in the NMR spectrum. These RDCs are relatively easily measured and represent an abundant source of highly precise information on the relative orientations of different internuclear 'bonds' within the molecule. In addition, RDCs exhibit sensitivity to molecular motions on the nsec-msec timescales, during which many functionally important motions occur. An ongoing effort is to develop effective experimental as well as analytical tools for the characterization of biomolecular motions at atomic resolution.
A more applied interest of the laboratory is the mechanism by which misfolded or unfolded proteins in the endoplasmic reticulum are targeted for degradation by the cytosolic ubiquitin-proteasome system. It is well established that attachment of a K48-linked polyubiquitin (polyUb) chain to a protein leads to degradation by the 26S proteasome. We are focused on understanding how an E2:E3 enzyme pair (Ube2g2:gp78) work together to assemble K48-linked polyUb chains and then attach them to the proper substrates. Of particular interest is how these K48-linked polyUb chains are assembled with high specificity and efficiency. The solution to this problem will, among other things, require an understanding of how gp78 allosterically activates Ube2g2's catalytic function and how multiple proteins interact with one another in a dynamic fashion during chain assembly.
Bocik, W.E., A. Sircar, J.J. Gray, and J.R. Tolman. (2011) Mechanism of polyubiquitin chain recognition by the human ubiquitin conjugation enzyme Ube2g2. J. Biol. Chem. 286:3981-3991.
Arbogast, L., A. Majumdar, and J.R. Tolman. (2010) HNCO-based measurement of one-bond amide 15N-1H couplings with optimized precision. J. Biomol. NMR 46:175-189.
Ju, T., W. Bocik, A. Majumdar, and J.R. Tolman. (2010) Solution structure and dynamics of human ubquitin conjugating enzyme Ube2g2. Proteins: Struct. Funct. Bioinfo. 78:1291-1301.
Tolman, J.R. (2009) Protein dynamics from disorder. Nature 459:1063-1064.
Ruan, K., K.B. Briggman, and J.R. Tolman. (2008) De novo determination of internuclear vector orientations from residual dipolar couplings measured in three independent alignment media. J. Biomol. NMR 41:61-76.
Tolman, J.R., and K. Ruan. (2006) NMR residual dipolar couplings as probes of biomolecular dynamics. Chem. Rev. 106:1720-1736.
Ruan, K., and J.R. Tolman. (2005) Composite alignment media for the measurement of independent sets of NMR residual dipolar couplings. J. Am. Chem. Soc. 127:15032-15033.
Briggman, K.B., and J.R. Tolman. (2003) De novo determination of bond orientations and order parameters from residual dipolar couplings with high accuracy. J. Am. Chem. Soc.125:10164-10165.
Tolman, J.R. (2002) A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecular NMR spectroscopy. J. Am. Chem. Soc. 124:12020-12030.
