The enzyme rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) offers and unusual opportunity to study and understand functionally important features of protein structure and its fluctuations. The proposed research is designed to exploit advances made during the previous grant period by using rhodanese as a model for the role of domain interactions and protein flexibility in catalysis and ligand interactions. The overall aims are to continue to pursue correlations between flexible conformational states of rhodanese and the catalytic events in which it participates. The suggested approach is to use substrates and inhibitors to produce stable species which approximate catalytic intermediates, and to study their conformations with chemical, physical and functional methods that are sensitive to structural dynamics. Fluorometry is a central technique because it represents a minimally interactive probe sensitive to subtle and dynamic changes. However it is vital that information from a variety of approaches be correlated with functional studies. A major goal will be to characterize the dynamic properties of rhodanese species with steady-state and nanosecond fluorescence methods using the intrinsic fluorophores as well as a variety of probes. In addition, corroborative techniques that can sense structural dynamics will be used, e.g., tritium exchange, rapid reaction kinetic methods and differential chemical modification. These results will be compared with these from methods that sense average structure, e.g., CD and ultracentrifugation. This information is then to be correlated with measured kinetic behavior and interpreted with the aid of a high resolution x-ray structure that is available. These approaches will be extended to understand the effects of low concentrations of denaturants, pH and ionic strength, all of which have particularly marked effects on both the structure and function of rhodanese. An underlying objective is to develop a set of techniques for the recognition and understanding of protein flexibility and domain interactions in a variety of other functional systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025177-10
Application #
3272826
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1978-04-01
Project End
1989-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Type
School of Medicine & Dentistry
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Panda, Markandeswar; Horowitz, Paul M (2004) Activation parameters for the spontaneous and pressure-induced phases of the dissociation of single-ring GroEL (SR1) chaperonin. Protein J 23:85-94
Panda, Markandeswar; Horowitz, Paul M (2002) Conformational heterogeneity is revealed in the dissociation of the oligomeric chaperonin GroEL by high hydrostatic pressure. Biochemistry 41:1869-76
Ramachandiran, Vasanthi; Kramer, Gisela; Horowitz, Paul M et al. (2002) Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro. FEBS Lett 512:209-12
Panda, Markandeswar; Ybarra, Jesse; Horowitz, Paul M (2002) Dissociation of the single-ring chaperonin GroEL by high hydrostatic pressure. Biochemistry 41:12843-9
Kramer, Gisela; Ramachandiran, Vasanthi; Horowitz, Paul M et al. (2002) The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor. Arch Biochem Biophys 403:63-70
Panda, M; Smoot, A L; Horowitz, P M (2001) The 4,4'-dipyridyl disulfide-induced formation of GroEL monomers is cooperative and leads to increased hydrophobic exposure. Biochemistry 40:10402-10
Smoot, A L; Panda, M; Brazil, B T et al. (2001) The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL. Biochemistry 40:4484-92
Panda, M; Ybarra, J; Horowitz, P M (2001) High hydrostatic pressure can probe the effects of functionally related ligands on the quaternary structures of the chaperonins GroEL and GroES. J Biol Chem 276:6253-9
Kramer, G; Ramachandiran, V; Horowitz, P et al. (2001) An additional serine residue at the C terminus of rhodanese destabilizes the enzyme. Arch Biochem Biophys 385:332-7
Nandi, D L; Horowitz, P M; Westley, J (2000) Rhodanese as a thioredoxin oxidase. Int J Biochem Cell Biol 32:465-73

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