Linked Life Data

2387847

Source:http://linkedlifedata.com/resource/pubmed/id/2387847

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
24
pubmed:dateCreated
1990-9-27
pubmed:databankReference
pubmed:abstractText
In principle, protein thermostability depends on efficient interior packing of apolar residues and on avoidance of irreversible denaturation in the unfolded state. To study these effects, the single free cysteine in the highly stable enzyme bovine Cu,Zn superoxide dismutase was mutated to alanine (Cys6----Ala), and the recombinant protein was expressed in yeast, purified, characterized for reversible and irreversible denaturation, crystallized isomorphously to the wild-type enzyme, and used to determine the atomic structure. Removal of the chemically reactive thiol significantly decreased the rate of irreversible denaturation (as monitored by thermal inactivation at 70 degrees C), but the observed energetic cost (delta delta G of 0.7-1.3 kcal/mol as determined by differential scanning calorimetry) was much less than predicted from either the change in hydrophobicity or packing due to removal of the interior sulfur atom. X-ray diffraction data were collected to 2.1-A resolution using an area detector, and the atomic model for the mutant enzyme was determined by fitting to electron density difference maps, followed by reciprocal space refinement both with stereochemical restraints using PROLSQ and with molecular dynamics using X-PLOR. The refined 2.1-A resolution crystallographic structure suggests that small concerted and compensating shifts (less than 0.5 A) of the surrounding side chains and of the adjacent N- and C-terminal beta-strands significantly reduced the energetic cost of the interior mutation by improving packing and stereochemistry in the mutant enzyme. Taken together, these results differentiate between the effects of reversible and irreversible processes as they impact the design of thermostable proteins and suggest that relatively subtle concerted shifts can significantly reduce the energetic cost of evolutionary variation in internal residues of proteins with Greek key beta-barrel folds.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
25
pubmed:volume
265
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
14234-41
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Changes in crystallographic structure and thermostability of a Cu,Zn superoxide dismutase mutant resulting from the removal of a buried cysteine.
pubmed:affiliation
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't