Detection of an intermediate in the folding of the (beta alpha)8-barrel N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli.


Abstract

We have used thermodynamic and kinetic techniques to monitor the guanidinium chloride induced (GdmCl-induced) denaturation of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli (ePRAI). Although CD-monitored equilibrium denaturation curves are consistent with cooperative unfolding of the protein centered at 1.45 M GdmCl, fluorescence readings drop by over 25% in the region preceding the CD-monitored transition, suggesting non-two-state behavior. Kinetics experiments measure a slow relaxation rate with negative fluorescence amplitude when protein is diluted from 0 to 0.5 M GdmCl, corroborating results from equilibrium conditions. Detection of several unfolding and refolding rates in final GdmCl concentrations from 0 to 5.0 M indicates the presence of at least one intermediate along unfolding and refolding pathways. GdmCl dependence of the relaxation rates can be explained most easily by a nonsequential mechanism for ePRAI unfolding, though a sequential mechanism cannot be ruled out. The data corroborate the fragment complementation studies of Eder and Kirschner [Eder, J., & Kischner, K. (1992) Biochemistry 31, 3617-3625], which are consistent with unfolding of the C-terminal portion of a yeast-derived PRAI in its folding intermediate. In ePRAI, such partial unfolding would expose W391 to quenching by solvent molecules; W356, ePRAI's other tryptophan, is buried in the hydrophobic core and is unlikely to be affected by local changes in structure. A C-terminally unfolded folding intermediate has been demonstrated in the folding of tryptophan synthase (alpha-subunit), a related beta alpha-barrel enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4465, 4466 Extra Details: cooperative unfolding; non-two-state behavior;,sequential mechanism; partial unfolding; beta alpha-barrel enzyme

Submission Details

ID: sdXU3RuJ4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Jasanoff A;Davis B;Fersht AR,Biochemistry (1994) Detection of an intermediate in the folding of the (beta alpha)8-barrel N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli. PMID:8193151
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