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Thermodynamics of Denaturation of Mutants of Barnase with Disulfide Crosslinks

Johnson, Christopher M; Oliveberg, Mikael LU ; Clarke, Jane and Fersht, Alan R (1997) In Journal of Molecular Biology 268(1). p.198-208
Abstract
We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for... (More)
We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (@DCpd), determined from the slope of the enthalpy of denaturation versus temperature, are not confined to mutants with disulfide crosslinks. The value of @DCpd is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disulfide crosslink, while the @DCpd remains unchanged in a destabilised mutant containing a disulfide. The variation in @DCpd may result from an inherent temperature-dependence of @DCpd, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disulfide mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93-R69 salt-bridge present in the wild-type. This finding confirms initial observations in the X-ray structure of this disulfide mutant that the salt-bridge has been disrupted by the introduced crosslink. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
thermodynamics, denaturation, entropy, folding, enthalpy
in
Journal of Molecular Biology
volume
268
issue
1
pages
198 - 208
publisher
Elsevier
external identifiers
  • scopus:0031585997
ISSN
1089-8638
DOI
10.1006/jmbi.1997.0928
language
English
LU publication?
yes
id
5ac35495-0367-46ef-aab6-1b3de8e7e46e (old id 126147)
date added to LUP
2007-07-05 16:16:26
date last changed
2017-05-14 04:14:41
@article{5ac35495-0367-46ef-aab6-1b3de8e7e46e,
  abstract     = {We have measured the effects of disulfide crosslinks on the thermodynamics of denaturation of three mutants of barnase that contain cystine and the corresponding single and double cysteine mutants. At first sight, the data are consistent with the hypothesis that disulfide crosslinks stabilise proteins through entropic destabilisation of the denatured state, but the decreases in the entropy of denaturation are larger than predicted and are accompanied by decreases in the enthalpy of denaturation. These effects are not a unique feature of the disulfide crosslink and are observed in a range of non-crosslinked mutants of barnase as part of a general enthalpy-entropy compensation phenomenon. Similarly, effects on the heat capacity change for denaturation (@DCpd), determined from the slope of the enthalpy of denaturation versus temperature, are not confined to mutants with disulfide crosslinks. The value of @DCpd is lower in four stabilised mutants than in wild-type barnase, irrespective of the presence of a disulfide crosslink, while the @DCpd remains unchanged in a destabilised mutant containing a disulfide. The variation in @DCpd may result from an inherent temperature-dependence of @DCpd, since it is measured for each mutant over a different temperature range. The thermodynamics of denaturation of the disulfide mutant with a crosslink between positions 70 and 92 change anomalously with pH but in a similar way to that of the D93N mutant of barnase, which lacks the D93-R69 salt-bridge present in the wild-type. This finding confirms initial observations in the X-ray structure of this disulfide mutant that the salt-bridge has been disrupted by the introduced crosslink.},
  author       = {Johnson, Christopher M and Oliveberg, Mikael and Clarke, Jane and Fersht, Alan R},
  issn         = {1089-8638},
  keyword      = {thermodynamics,denaturation,entropy,folding,enthalpy},
  language     = {eng},
  number       = {1},
  pages        = {198--208},
  publisher    = {Elsevier},
  series       = {Journal of Molecular Biology},
  title        = {Thermodynamics of Denaturation of Mutants of Barnase with Disulfide Crosslinks},
  url          = {http://dx.doi.org/10.1006/jmbi.1997.0928},
  volume       = {268},
  year         = {1997},
}