Abstract

Methodology for observation of the kinetics of the refolding of denatured proteins into their native conformation will be developed. The decrease in the effective hydrodynamic volume will be observed using exclusion chromatography at high pressure. The length of the column, the resiliance of the matrix, and the geometry of the chromatographic assembly will be optimized to facilitate measurement of the kinetic parameters typically observed in the folding of small proteins at low temperature. A stopped-flow circular dichroism in instrument will be tested and modified to observe the formation of secondary structural elements and their assembly. A rapid scan stopped-flow absorbance and fluorescence spectrometer will also be tested to observe changes in the environments of intrinsic and extrinsic chromophores during folding. Studies of the mechanism of the folding of a protein containing the dinucleotide fold, thioredoxin, and variants thereof will be continued. The structure of a stable folding intermediate will be examined in detail. The equilibrium and kinetic parameters of the cis/trans isomerization of proline 76 will be measured. Additional intermediate forms will be searched for by multimixing protocols. The stability and folding of the individual globular domains will be examined as well as the role of their association in the folding mechanism. Variants of the protein will be constructed by site specitic mutations to perturb aspects of the folding mechanism.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022109-16
Application #
3270939
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1981-07-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1993-03-31
Support Year
16
Fiscal Year
1991
Total Cost
Indirect Cost

  Institution

Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242

  Publications

Park, S H; Shalongo, W; Stellwagen, E (1997) The role of PII conformations in the calculation of peptide fractional helix content. Protein Sci 6:1694-700
Park, S H; Shalongo, W; Stellwagen, E (1993) Residue helix parameters obtained from dichroic analysis of peptides of defined sequence. Biochemistry 32:7048-53
Kopperschlager, G; Bar, J; Stellwagen, E (1993) Limited proteolysis of yeast phosphofructokinase. Sequence locations of cleavage sites created by the actions of different proteinases. Eur J Biochem 217:527-33
Shalongo, W; Heid, P; Stellwagen, E (1993) Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 1. Denaturant dependent baseline changes. Biopolymers 33:127-34
Shalongo, W; Jagannadham, M; Stellwagen, E (1993) A reexamination of the conformational transitions of T4 thioredoxin. Biopolymers 33:903-13
Park, S H; Shalongo, W; Stellwagen, E (1993) Modulation of the helical stability of a model peptide by ionic residues. Biochemistry 32:12901-5
Shalongo, W; Jagannadham, M; Stellwagen, E (1993) Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 2. Comparison of spectral and chromatographic kinetic analyses. Biopolymers 33:135-45
Shalongo, W; Jagannadham, M V; Heid, P et al. (1992) A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography. Biochemistry 31:11390-6
Shalongo, W; Jagannadham, M V; Flynn, C et al. (1989) Refolding of denatured ribonuclease observed by size exclusion chromatography. Biochemistry 28:4820-5
Kelley, R F; Shalongo, W; Jagannadham, M V et al. (1987) Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin. Biochemistry 26:1406-11

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