Linked Life Data

15035606

Source:http://linkedlifedata.com/resource/pubmed/id/15035606

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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
2004-3-23
pubmed:abstractText
The nature of the rate-limiting transition state at zero denaturant (TS(1)) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS(1) has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degrees C and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS(2)). Therefore, it exists after TS(2) in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rd-apocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
30
pubmed:volume
43
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3346-56
pubmed:meshHeading
pubmed-meshheading:15035606-Alanine, pubmed-meshheading:15035606-Amino Acid Substitution, pubmed-meshheading:15035606-Bacillus, pubmed-meshheading:15035606-Deuterium Exchange Measurement, pubmed-meshheading:15035606-Glycine, pubmed-meshheading:15035606-Guanidine, pubmed-meshheading:15035606-Hydrogen-Ion Concentration, pubmed-meshheading:15035606-Kinetics, pubmed-meshheading:15035606-Models, Chemical, pubmed-meshheading:15035606-Nonlinear Dynamics, pubmed-meshheading:15035606-Nuclear Magnetic Resonance, Biomolecular, pubmed-meshheading:15035606-Protein Conformation, pubmed-meshheading:15035606-Protein Denaturation, pubmed-meshheading:15035606-Protein Folding, pubmed-meshheading:15035606-Protein Structure, Secondary, pubmed-meshheading:15035606-Protons, pubmed-meshheading:15035606-Ribonucleases, pubmed-meshheading:15035606-Signal Transduction
pubmed:year
2004
pubmed:articleTitle
The folding pathway of barnase: the rate-limiting transition state and a hidden intermediate under native conditions.
pubmed:affiliation
Laboratory of Biochemistry, National Cancer Institute, NIH, Building 37, Room 6114E, Bethesda, Maryland 20892, USA.
pubmed:publicationType
Journal Article