BIOCHEMISTRY AND BIOPHYSICS OF BPTI FOLDING MUTANTS

Project Details

Description

The broad, long-term objective of this work is to elucidate the rules that govern protein folding. Bovine pancreatic trypsin inhibitor (BPTI) was chosen as a model system because it is a simple globular polypeptide and a large amount of structural, biophysical, and theoretical data is already available for the wild type protein. BPTI is also the best understood member of the Kunitz class of protease inhibitors, a family that includes several members relevant to human physiology and disease including the lipoprotein-associated coagulation inhibitor and the alternatively-spliced domain of the Alzheimer's amyloid beta protein precursor. The overall basic thrust of the research has been to make genetically-modified forms of BPTI that have perturbed folding properties, express these proteins in quantity, and characterize them biophysically. The specific research priorities for this grant are: 1) Address the problem of "designing out" disulfide bonds by making BPTI mutants missing two or more of the native disulfides; 2) Conduct a quantitative survey of all stabilizing interactions in the molecule by systematically mutating each non-alanine residue to alanine and characterizing the stabilization free energies of the resulting mutants; 3) Test the "cardboard box" model of protein folding by measuring the contribution of electrostatics to 5-55 disulfide bond formation and to global polypeptide stability; 4) Facilitate ongoing work in collaborators' laboratories aimed at investigating the structure and dynamics of selected BPTI mutants. Presumably, this combined genetic and biophysical approach to the dissection of the folding mechanism of a very simple model protein will illuminate the still poorly-understood process of protein folding in general.
StatusFinished
Effective start/end date7/1/916/30/94

Funding

  • National Institute on Aging

ASJC

  • Structural Biology
  • Biochemistry

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