Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/9345
Share/Export:
logo share SHARE logo core CORE BASE
Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL | DATACITE
Title

Determining denaturation midpoints in multiprobe equilibrium protein folding experiments

AuthorsNaganathan, Athi N. CSIC ORCID; Muñoz, Víctor
Issue Date10-Jun-2008
PublisherAmerican Chemical Society
CitationBiochemistry 47(26): 6752-6761 (2008)
AbstractMultiprobe equilibrium unfolding experiments in the downhill regime (i.e., maximal barrier < 3RT) can resolve the folding process with atomic resolution [Munoz (2002) Int. J. Quantum Chem. 90, 1522-1528]. Such information is extracted from hundreds of heterogeneous atomic equilibrium unfolding curves, which are characterized according to their denaturation midpoint (e.g., Tm for thermal denaturation). Using statistical methods, we analyze Tm accuracy when determined from the extremum of the derivative of the unfolding curve and from two-state fits under different sets of simulated experimental conditions. We develop simple procedures to discriminate between real unfolding heterogeneity at the atomic level and experimental uncertainty in the single Tm of conventional two-state folding. We apply these procedures to the recently published multiprobe NMR experiments of BBL [Sadqi et al. (2006) Nature 442, 317-321] and conclude that for the 122 single transition atomic unfolding curves reported for this protein the mean Tm accuracy is better than 1.8 K for both methods, compared to the 60 K spread in Tm determined experimentally. Importantly, we also find that when the pre- or posttransition baseline is incomplete, the two-state fits systematically drift the estimated Tm value toward the center of the experimental range. Therefore, the reported 60 K Tm spread in BBL is in fact a lower limit. The derivative method is significantly less sensitive to this problem and thus is a better choice for multiprobe experiments with a broad Tm distribution. The results we obtain in this work lay the foundations for the quantitative analysis of future multiprobe unfolding experiments in fast-folding proteins.
Description10 pages, 7 figures.-- PMID: 18540681 [PubMed].
Publisher version (URL)http://dx.doi.org/10.1021/bi800336x
URIhttp://hdl.handle.net/10261/9345
DOI10.1021/bi800336x
ISSN0006-2960
Appears in Collections:(CIB) Artículos

Show full item record
Review this work

SCOPUSTM   
Citations

23
checked on May 13, 2022

WEB OF SCIENCETM
Citations

21
checked on May 14, 2022

Page view(s)

338
checked on May 16, 2022

Google ScholarTM

Check

Altmetric

Dimensions


WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.