Recent work has shown that proteins under extreme denaturing conditions retain a net alignment with respect to an NMR magnetic field when immersed in the asymmetric environment of liquid crystals or gels. The asymmetric shapes of these matrices impose weak steric restrictions on the molecular reorientation of solutes. The resulting time- and ensemble-averaged alignment leads to incomplete cancellation of NMR dipolar couplings. Dipolar couplings report on the orientation of individual bond vectors with respect to the molecular alignment frame. The objectives of this project are to further the understanding of the structural basis for net alignment in denatured proteins and to use the alignment information encoded in dipolar couplings towards a structural characterization of denatured states. The objectives will be addressed with two model systems. The first consists of the two proteins CspA and LysN, which share a native five-stranded beta-barrel structural motif but no detectable sequence similarity. Dipolar couplings will be used to investigate the extent to which native-like structure is conserved between the denatured states of the two proteins. The second model system is comprised of two polypeptides that adopt regular extended alpha-helical structures: the S-peptide, which corresponds to the first alpha-helix of the protein ribonuclease A; and the GCN4-p alpha-helical coiled-coil dimer. Dipolar couplings will be used to determine the persistence length of alpha-helical conformations in the denatured forms of the two polypeptides.
The principal motivation of this work is to obtain an improved understanding of the structural basis of protein folding. Protein folding remains one of the most difficult and intriguing problems in biology today, and is essential for progress in structural genomics. The new experimental window on structure in denatured states afforded by NMR alignment data, will allow more accurate modeling of protein folding and of the factors responsible for the stability of proteins to unfolding. Modeling of protein folding requires information on denatured states in as much as structure in these initial states can favor or oppose folding to the functional native state. The structural characterization of denatured states additionally impacts mechanisms for denatured protein aggregation reactions, which can occur through illegitimate recombination of incompletely formed nascent structures. The delineation of structural building blocks that persist in denatured proteins provides insights into the mechanisms by which protein structures evolve, information that in turn could be useful in protein design. This project offers the opportunity for students to train in a rich, multidisciplinary approach to protein folding that combines protein chemistry, state of the art NMR experiments, and structure modeling.