The goal of this research is to understand the mechanisms by which a protein folds spontaneously to a unique three-dimensional conformation. This research project will exploit the fact that all the information required for folding is contained in the amino acid sequence, by studying the folding behavior of proteins whose sequence has been altered by missense mutation. The system to be studied is a bacterial protein, the Alpha subunit of tryptophan synthase. A large number of strains of Escherichia coli with missense mutations in the Alpha gene region of the tryptophan operon have been isolated and the amino acid substitution in the Alpha subunit identified. In vitro equilibrium and kinetic studies of the reversible unfolding transition of wild-type and mutant proteins will monitor unfolding by changes in optical properties and by calorimetry. Site-directed mutagenesis will also be used to introduce amino acid replacements at new positions in the sequence. The results will be interpreted in terms of a folding model for the Alpha subunit, developed in our laboratory, which will allow quantitative comparison of the effect of the amino acid replacement. From this comparison, it will be possible to learn which amino acids play a key role in the folding process. These conclusions will then be combined with structural information from x-ray studies and predictive schemes in order to understand the effects of the mutation at a molecular level. Knowledge of the involvement of these key amino acids in stabilizing secondary and tertiary structures in the native, folded conformation will permit an assessment of those features that are critical to folding. This information will be useful in predicting the tertiary structure from amino acid sequence, in understanding the molecular basis of inherited diseases and in the design of new enzyme catalysts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023303-14
Application #
3271588
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1976-05-01
Project End
1990-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
14
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
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
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

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