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
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3KZ3 2010-02-23 1.64 A structure of a lambda repressor fragment mutant
1LMB 1991-11-05 1.8 REFINED 1.8 ANGSTROM CRYSTAL STRUCTURE OF THE LAMBDA REPRESSOR-OPERATOR COMPLEX
5ZCA 2018-08-15 1.8 Crystal structure of lambda repressor (1-20) fused with maltose-binding protein
1F39 2000-07-26 1.9 CRYSTAL STRUCTURE OF THE LAMBDA REPRESSOR C-TERMINAL DOMAIN
3WOA 2015-04-29 2.0 Crystal structure of lambda repressor (1-45) fused with maltose-binding protein
1LLI 1994-08-31 2.1 THE CRYSTAL STRUCTURE OF A MUTANT PROTEIN WITH ALTERED BUT IMPROVED HYDROPHOBIC CORE PACKING
1RIO 2004-01-27 2.3 Structure of bacteriophage lambda cI-NTD in complex with sigma-region4 of Thermus aquaticus bound to DNA
1KCA 2001-12-21 2.91 Crystal Structure of the lambda Repressor C-terminal Domain Octamer
1LRP 1989-01-09 3.2 COMPARISON OF THE STRUCTURES OF CRO AND LAMBDA REPRESSOR PROTEINS FROM BACTERIOPHAGE LAMBDA
3BDN 2008-04-15 3.91 Crystal Structure of the Lambda Repressor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Repressor protein cI P03034 RPC1_LAMBD