Document Detail


Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase.
MedLine Citation:
PMID:  7499309     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.
Authors:
S G Choi; J K Hardman
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Publication Detail:
Type:  Comparative Study; Journal Article    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  270     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1995 Nov 
Date Detail:
Created Date:  1996-01-17     Completed Date:  1996-01-17     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  28177-82     Citation Subset:  IM    
Affiliation:
Department of Biological Sciences, University of Alabama, Tuscaloosa 35487, USA.
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MeSH Terms
Descriptor/Qualifier:
Calorimetry
Escherichia coli / enzymology*
Kinetics
Luminescent Measurements
Macromolecular Substances
Models, Structural
Mutagenesis, Site-Directed
Point Mutation
Protein Denaturation
Protein Folding
Recombinant Proteins / chemistry,  drug effects,  isolation & purification
Spectrometry, Fluorescence
Tryptophan*
Tryptophan Synthase / chemistry*,  drug effects,  isolation & purification
Urea / pharmacology
Chemical
Reg. No./Substance:
0/Macromolecular Substances; 0/Recombinant Proteins; 57-13-6/Urea; 73-22-3/Tryptophan; EC 4.2.1.20/Tryptophan Synthase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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