A molecular-level understanding of the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to its native, functional conformation remains as one of the outstanding challenges in molecular biophysics. Although computer simulations are successfully folding small proteins and domains, the precise role of the amino acid sequence in defining the structures and stabilities of the species that populate the folding free energy surface remains elusive. We hypothesize that clusters of branched aliphatic side chains, isoleucines, leucines and valines (ILV), serve as cores of stability in folding intermediates and the native states of TIM barrel proteins, one of the most common motifs in biology. We propose a multi- faceted test of this hypothesis on a trio of indole-3-glycerolphosphate synthase (IGPS) orthologs, whose low sequence identity results in varying sizes and locations of their resident ILV clusters. A battery of techniques will probe the structures of partially-folded states that populate the folding free energy surfaces and test their relationship with these saturated hydrocarbon clusters. CD, FRET and SAXS techniques will assess secondary structure and provide pair-wise and global dimensional information beginning in the microsecond time range, hydrogen-exchange mass spectrometry and NMR methods will map the stable hydrogen bonding networks in partially-folded states that appear in the milliseconds-to-seconds time frame, and side chain burial in these same species will be assessed with a novel oxidative labeling method. The results will be used to validate the predictions of structure in partially-folded states using native-centric GM-model simulations that are capable of defining the entire folding reaction coordinate of these orthologs. In an exciting new venture, we will assess the effects of an exhaustive set of amino acid replacements in all 8 ?-strand and preceding ?/? loop stability elements on the relative fitness of each ortholog in a growth competition assay in a yeast strain lacking its intrinsic IGPS gene. The presumption that fitness provides an in vivo estimate of stability will be validated with an in vitro quantitative assessment of the perturbation of the stability of native and intermediate states in a subset of ~100 site-directed mutations in the same stability elements in the SsIGPS ortholog. Parallel CD and enzymatic activity assays will measure the effects of the mutations on the structure and the function of the enzyme as an alternative explanation for decrease in fitness. The output of these in vivo and in vitro measures of stability perturbations will serve as input for a bioinformatics analysis designed to offer a statistically- significant assessment of the context dependence of the mutations. Comparisons of the effects of mutations within and external to ILV clusters will provide an unbiased and robust approach towards determining their significance in defining cores of stability in globular proteins. Validation of our hypothesis, that clusters of branched aliphatic side chains play crucial roles in stabilizing partially-folded states and guiding the folding of TIM barrel proteins, has the potential to have a very broad impact on biology, biotechnology and medicine.
Understanding the process by which the 1 dimensional amino acid sequence of a protein is decoded into its functional 3 dimensional structure would be a fundamental breakthrough in biology and provide a rational basis for protein reengineering and de novo design in biotechnology. A deeper understanding of the role of mutations in destabilizing the native, functional conformations of proteins and, thereby, increasing the population of aggregation-prone partially-folded states would provide valuable insights into the molecular mechanisms of a host of human diseases.
|Watson, Matthew D; Peran, Ivan; Zou, Junjie et al. (2017) Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure. Biochemistry 56:1085-1094|
|Kathuria, Sagar V; Chan, Yvonne H; Nobrega, R Paul et al. (2016) Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability. Protein Sci 25:662-75|
|Peran, Ivan; Watson, Matthew D; Bilsel, Osman et al. (2016) Selenomethionine, p-cyanophenylalanine pairs provide a convenient, sensitive, non-perturbing fluorescent probe of local helical structure. Chem Commun (Camb) 52:2055-8|
|Rosen, Laura E; Kathuria, Sagar V; Matthews, C Robert et al. (2015) Non-native structure appears in microseconds during the folding of E. coli RNase H. J Mol Biol 427:443-53|
|Zhou, Huan-Xiang; Bilsel, Osman (2014) SAXS/SANS probe of intermolecular interactions in concentrated protein solutions. Biophys J 106:771-3|
|Kathuria, Sagar V; Kayatekin, Can; Barrea, Raul et al. (2014) Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS. J Mol Biol 426:1980-94|
|Kathuria, Sagar V; Chan, Alexander; Graceffa, Rita et al. (2013) Advances in turbulent mixing techniques to study microsecond protein folding reactions. Biopolymers 99:888-96|
|Gangadhara, Basavanapura N; Laine, Jennifer M; Kathuria, Sagar V et al. (2013) Clusters of branched aliphatic side chains serve as cores of stability in the native state of the HisF TIM barrel protein. J Mol Biol 425:1065-81|
|Graceffa, Rita; Nobrega, R Paul; Barrea, Raul A et al. (2013) Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam. J Synchrotron Radiat 20:820-5|
|Das, Payel; Kapoor, Divya; Halloran, Kevin T et al. (2013) Interplay between drying and stability of a TIM barrel protein: a combined simulation-experimental study. J Am Chem Soc 135:1882-90|
Showing the most recent 10 out of 61 publications