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 Institutes of Health: $141,720.00
  • National Institutes of Health
  • National Institutes of Health: $134,526.00

ASJC

  • Medicine(all)

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