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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM023303-37A1
Application #
8632260
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1976-05-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
37
Fiscal Year
2014
Total Cost
$381,163
Indirect Cost
$152,922
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
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
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
Zhou, Huan-Xiang; Bilsel, Osman (2014) SAXS/SANS probe of intermolecular interactions in concentrated protein solutions. Biophys J 106:771-3
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
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
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
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
Kathuria, Sagar V; Guo, Liang; Graceffa, Rita et al. (2011) Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering. Biopolymers 95:550-8
Yang, Xiaoyan; Kathuria, Sagar V; Vadrevu, Ramakrishna et al. (2009) Betaalpha-hairpin clamps brace betaalphabeta modules and can make substantive contributions to the stability of TIM barrel proteins. PLoS One 4:e7179
Wu, Ying; Kondrashkina, Elena; Kayatekin, Can et al. (2008) Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein. Proc Natl Acad Sci U S A 105:13367-72

Showing the most recent 10 out of 57 publications