A Comparison of the pH, Urea, and Temperature-denatured States of Barnase by Heteronuclear NMR: Implications for the Initiation of Protein Folding

A Comparison of the pH, Urea, and Temperature-denatured States of Barnase by Heteronuclear NMR:... The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile→Val51) which has an appropriate rate of interconversion between native and denatured states in urea. 3 J NH-C α H coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of β-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the β-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding process. Thus, studies of the structure of denatured states pinpoint where nucleation sites may be, and the kinetic and protein engineering studies show which ones are productive. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

A Comparison of the pH, Urea, and Temperature-denatured States of Barnase by Heteronuclear NMR: Implications for the Initiation of Protein Folding

Loading next page...
 
/lp/elsevier/a-comparison-of-the-ph-urea-and-temperature-denatured-states-of-C52gPlJhBb
Publisher
Elsevier
Copyright
Copyright © 1995 Academic Press
ISSN
0022-2836
DOI
10.1006/jmbi.1995.0618
Publisher site
See Article on Publisher Site

Abstract

The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile→Val51) which has an appropriate rate of interconversion between native and denatured states in urea. 3 J NH-C α H coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of β-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the β-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding process. Thus, studies of the structure of denatured states pinpoint where nucleation sites may be, and the kinetic and protein engineering studies show which ones are productive.

Journal

Journal of Molecular BiologyElsevier

Published: Nov 24, 1995

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create folders to
organize your research

Export folders, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off