# SYNTHETIC STUDIES OF PROTEIN STABILITY AND FOLDING

> **NIH NIH R01** · UNIVERSITY OF MINNESOTA · 1994 · $176,516

## Abstract

This research program applies state-of-the-art mild chemical methods for
peptide synthesis to prepare analogues of bovine pancreatic trypsin
inhibitor (BPTI), which are then studied by a series of biophysical
techniques in order to learn about structure, dynamics, and folding. BPTI
is a small 58-residue protein with an array of three disulfide bridges.
Extensive preliminary results have demonstrated the syntheses and
characterizations of BPTI with the natural sequence, and of two analogues
which replace paired half-cystine residues by paired alpha-amino-n-butyric
acid (Abu) isosteres. It has been shown that whereas BPTI with all six
half-cystines replaced by Abu (all-Abu-BPTI) behaves as a true random
coil, BPTI with one intact disulfide between half-cystines 14 and 38
([Cys14,38]-BPTI) is partially folded, with native-like structure in the
hydrophobic core and disordered structure in the rest of the molecule.
[Cys14,38]-BPTI represents a unique example of a protein which forms both
a "molten globule" and an "A state;" these species have structures very
similar to the transient intermediate formed in the first 10 to 20 msec of
the protein folding process. Specific aims of this research are two-fold,
seeking to synthesize and characterize proteins that are partially folded,
and conversely, to synthesize and characterize circularized or circular
permuted variants that may have enhanced stability. Proposed BPTI
analogues may contain in a single molecule those sequences that encode
initiation of folding and the final tertiary fold, for example by
circularizing all-Abu-BPTI with a peptide bond connecting the N- and C-
termini [these are proximal in native BPTI], by using oligoglycyl (or
related) spacers to connect BPTI sequences corresponding to the slow-
exchange core, or by substituting key residues in the core of [Cys14,38]-
BPTI. The apparent "circularity" of BPTI, and the role of disulfide
bridges in governing the protein's stability and postulated folding
pathways, will be tested by syntheses of parent molecules and analogues of
circularly permuted and circular BPTI' s with one or more disulfide
bridges replaced by Abu residues, and/or side-chain lactam, thioether
(carba), or dicarba bridges. Questions to be asked include: How does the
nature of amino acids in the hydrophobic core affect its formation? How
does the order of the secondary structural elements affect the
stabilities, structures, and activities of the proteins? How does the
order of the cysteine residues affect the acquisitions and stabilities of
final conformations? What are the minimal covalent constraints required to
achieve native-like conformations and activities? Can additional covalent
constraints lead to more stable proteins? These studies represent a new
approach to BPTI folding, and are expected to lead to significant insights
that contribute to the "protein folding problem." A further goal of this
work is to develop a general methodology for stabilizing therapeutically
pr...

## Key facts

- **NIH application ID:** 2190291
- **Project number:** 1R01GM051628-01
- **Recipient organization:** UNIVERSITY OF MINNESOTA
- **SAM.gov UEI:** KABJZBBJ4B54
- **Principal Investigator:** George Barany
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1994
- **Award amount:** $176,516
- **Award type:** 1
- **Project period:** 1994-08-01 → 1998-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/2190291

## Citation

> US National Institutes of Health, RePORTER application 2190291, SYNTHETIC STUDIES OF PROTEIN STABILITY AND FOLDING (1R01GM051628-01). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/2190291. Licensed CC0.

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