Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding.


Abstract

The absence of equilibrium intermediates in protein folding reactions (i.e., two-state folding) simplifies thermodynamic and kinetic analyses but is difficult to prove rigorously. We demonstrate a sensitive method for detecting partially folded species based on using proton chemical shifts as local probes of structure. The coincidence of denaturation curves for probes throughout the molecule is a particularly stringent test for two-state folding. In this study we investigate a new form of the N-terminal domain of bacteriophage lambda repressor consisting of residues 6-85 (lambda 6-85) using nuclear magnetic resonance (NMR) and circular dichroism (CD). This truncated version lacks the residues required for dimerization and is monomeric under the conditions used for NMR. Heteronuclear NMR was used to assign the 1H, 15N, and backbone 13C resonances. The secondary and tertiary structure of lambda 6-85 is very similar to that reported for the crystal structure of the DNA-bound 1-92 fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196], as judged by analysis of chemical shifts, amide hydrogen exchange, amide-alpha coupling constants, and nuclear Overhauser enhancements. Thermal and urea denaturation studies were conducted using the chemical shifts of the four aromatic side chains as local probes and the CD signal at 222 nm as a global probe. Plots of the fraction denatured versus denaturant concentration obtained from these studies are identical for all probes under all conditions studied. This observation provides strong evidence for two-state folding, indicating that there are no populated intermediates in the folding of lambda 6-85. Study holds ProTherm entries: 4673 Extra Details: NaN3(1 mM) was added in the experiment partially folded species; two-state folding;,amide hydrogen exchange; chemical shifts; aromatic side chains

Submission Details

ID: o2w9QpcB3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Huang GS;Oas TG,Biochemistry (1995) Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding. PMID:7696251
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