Linked Life Data

8035461

Source:http://linkedlifedata.com/resource/pubmed/id/8035461

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
1994-8-18
pubmed:abstractText
Ribose-binding protein (RBP) consists of two alpha/beta globular units; the N domain being composed of four helices (I, II, III and IX) and six sheets (A, B, C, D, E and K), and the C domain with five helices (IV, V, VI, VII and VIII) and six sheets (F, G, H, I, J and L). The two domains are connected by three strands. In the previous study, tyrosine residues in the RBP were substituted by phenylalanine to examine the fluorescence property of each chromophore. Since the three tyrosine residues are scattered in the two domains of RBP, residues 32 and 261 in the N domain and 115 in the C domain, we were able to monitor the state of protein folding using unaltered tyrosine as a local probe. The final structures of the mutant proteins show little differences from those of wild-type as judged by circular dichroism spectra. The equilibrium and kinetic folding behaviors of the mutant RBPs were examined by fluorescence spectroscopy. The equilibrium data obtained from the mutant RBPs conform to the two-state transition involving the native and unfolded species. However, kinetic studies indicate that there exists an intermediate formed transiently during the folding or unfolding process, which was not detected in equilibrium experiments. In unfolding kinetics of the mutant protein retaining only the N domain tyrosine residues, a striking change in fluorescence was observed as an initial jump, suggesting the presence of a partial unfolding step of RBP around the Tyr32 chromophore, since the fluorescence of Tyr261 does not change upon folding. This occurred immediately after mixing with 0.6 to 1 M range of guanidine hydrochloride and was completed in less than three seconds of the mixing dead time. The partial increase of fluorescence appears to be due to the dissociation of a quenching group, the carboxyl side-chain of Asp249 located on helix IX, from the Tyr32 fluorophore. The successive unfolding process reflects a process of release from the second quenching group at Asp2 on the sheet A. The substitution at Tyr261 on sheet K by phenylalanine reveals an additional kinetic phase in refolding, in which the folding from unfolded into an intermediate form seems similar to wild-type in time scale, whereas the next step leading to the formation of native protein becomes slower.(ABSTRACT TRUNCATED AT 400 WORDS)
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0022-2836
pubmed:author
pubmed:issnType
Print
pubmed:day
22
pubmed:volume
240
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
385-95
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Engineered tyrosine residues serve as the local probes to detect a kinetic intermediate in the folding of ribose-binding protein.
pubmed:affiliation
Department of Life Science, Korea Advanced Institute of Science and Technology, Taejon.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't