Kevin H. Gardner
Structural Basis of Protein-Protein Interactions, Studied by NMR Methods
My laboratory uses solution nuclear magnetic resonance (NMR) methods to study the structures and dynamics of protein/protein complexes involved in regulating eukaryotic transcription and other biological processes.
Studies such as these have been made possible by the rapid evolution of NMR-based tools to study macromolecular structure. Until recently, NMR methods were successful at providing structures of only relatively small proteins smaller than 10-15 kDa molecular weight. However, the symbiotic development of stable isotope labeling, multidimensional NMR pulse sequence and computational methods now enables solution NMR to be used to provide high-resolution structures of 30-40 kDa protein systems and useful structural information on even larger proteins and their complexes. Given the focus of my research group on investigating macromolecular assemblies involved in complex regulatory processes, this increased molecular weight "limit" opens up a wider range of systems that are amenable to NMR-based analyses. In my research group, we will continue to develop and apply such novel methods to systems of biological interest.
As mentioned above, stable isotope labeling methodology has been an area of particularly active development. The success of NMR as a structural tool has been based in part on the facile production of macromolecular samples uniformly labeled with the spin-1/2 NMR-active isotopes of carbon (13C) and nitrogen (15N). With these isotopes in place, one can use sophisticated triple resonance experiments to generate the sequence-specific 13C, 15N, and 1H chemical shift assignments that provide the foundation for later analyses of macromolecular structure and dynamics. These methods are quite useful for studying systems under 25 kDa molecular weight, but their application on larger macromolecules has been hindered by broad peak linewidths that result in spectral overlap and poor sensitivity.
One approach to surpassing this limit has been the use of labeling schemes to produce proteins labeled with deuterium (2H) at all aliphatic positions in order to eliminate dipolar 13C-1H couplings that contribute to line broadening. Using such 15N, 13C, 2H labeled proteins, I have been able to assign the backbone and sidechain chemical shifts of a 42 kDa maltose binding protein (MBP)/carbohydrate complex (Gardner et al., 1998); similar approaches have also been successful with a 64 kDa trpR/DNA complex (Shan et al., 1996). Unfortunately, while such deuteration improves the sensitivity of many experiments, it also drastically reduces the number of interproton NOE-based distance restraints one can measure among the remaining solvent-exchangeable protons. As these restraints constitute the majority of experimental data used to generate NMR-derived structures, reducing their number and diversity significantly worsens the precision and accuracy of structures available from fully deuterated proteins (Gardner et al., 1997).
As such, I have developed chemical and biochemical approaches to generate highly deuterated proteins that contain a small number of highly protonated sites, typically at the methyl groups of Val, Leu and Ile (Gardner et al., 1996; Gardner & Kay, 1997). Proteins produced in this manner retain many of the benefits of deuteration, while methyl-amide and methyl-methyl NOEs provide an ensemble of distance restraints significantly larger than previously available, allowing the generation of medium resolution structures of proteins over 40 kDa using exclusively NMR-based data (Gardner et al., 1997).