Subunit dissociation affects DNA binding in a dimeric lac repressor produced by C-terminal deletion.

Abstract

The reduction in apparent operator binding affinity found for dimeric lac repressor proteins produced by disruption of the C-terminal coiled-coil interaction has been proposed to derive from thermodynamic linkage between dimer-monomer and protein-DNA equilibria [Brenowitz et al. (1991) J. Biol. Chem. 266, 1281]. To explore this linkage, we have employed two dimeric proteins, a deletion mutant (-11 aa) missing 11 amino acids at the C-terminus that has diminished apparent operator binding affinity [Chen & Matthews (1992) J. Biol. Chem. 267, 13843] and a mutant (R3) that binds to operator with wild-type affinity in which the C-terminal leucine heptad repeats of lac repressor were replaced by the GCN4 dimerization sequence [Alberti et al. (1993) EMBO J. 12, 3227; Chen et al. (1994) J. Biol. Chem. (in press)]. To avoid the complexities of working at the low concentrations of protein required by the high affinity between the monomer subunits, urea denaturation studies were undertaken to determine the free energy change(s) for dissociation and/or unfolding. Under denaturing conditions, dimer dissociation and monomer unfolding were found to be concerted processes, and the free energy change for the overall process of dimer to unfolded monomer was derived from these experiments for the two dimeric proteins. A monomeric mutant (Y282D) of the lactose repressor was examined to determine the free energy change of protomer unfolding. From the combination of these data, the Kd for -11 aa dimer dissociation was determined to be 7.7 x 10(-8) M, and the corresponding value for R3 protein was 3.2 x 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4470 Extra Details: operator binding affinity; coiled-coil interaction;,GCN4 dimerization sequence; genetic regulatory proteins

Submission Details

ID: aoDXnWZ6

Submitter: Connie Wang

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

Version: 1

Publication Details
Chen J;Matthews KS,Biochemistry (1994) Subunit dissociation affects DNA binding in a dimeric lac repressor produced by C-terminal deletion. PMID:8038163
Additional Information

Study Summary

Number of data points 1
Proteins Lactose operon repressor ; Lactose operon repressor
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dG_H2O
Libraries Mutations for sequence MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKTAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ

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
1LCD 1994-01-31 STRUCTURE OF THE COMPLEX OF LAC REPRESSOR HEADPIECE AND AN 11 BASE-PAIR HALF-OPERATOR DETERMINED BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND RESTRAINED MOLECULAR DYNAMICS
1LCC 1994-01-31 STRUCTURE OF THE COMPLEX OF LAC REPRESSOR HEADPIECE AND AN 11 BASE-PAIR HALF-OPERATOR DETERMINED BY NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND RESTRAINED MOLECULAR DYNAMICS
2BJC 2005-10-18 NMR structure of a protein-DNA complex of an altered specificity mutant of the lac repressor headpiece that mimics the gal repressor
2KEI 2009-05-19 Refined Solution Structure of a Dimer of LAC repressor DNA-Binding domain complexed to its natural operator O1
2KEJ 2009-05-19 Solution structure of a dimer of LAC repressor DNA-binding domain complexed to its natural operator O2
1OSL 2004-05-04 Solution structure of a dimeric lactose DNA-binding domain complexed to a nonspecific DNA sequence
1CJG 2000-01-01 NMR STRUCTURE OF LAC REPRESSOR HP62-DNA COMPLEX
2KEK 2009-05-19 Solution structure of a dimer of LAC repressor DNA-binding domain complexed to its natural operator O3
1LQC 1997-02-12 LAC REPRESSOR HEADPIECE (RESIDUES 1-56), NMR, 32 STRUCTURES
1L1M 2002-06-26 SOLUTION STRUCTURE OF A DIMER OF LAC REPRESSOR DNA-BINDING DOMAIN COMPLEXED TO ITS NATURAL OPERATOR O1
1JYE 2001-10-18 1.7 Structure of a Dimeric Lac Repressor with C-terminal Deletion and K84L Substitution
2P9H 2007-06-19 2.0 High resolution structure of the Lactose Repressor bound to IPTG
3EDC 2008-11-25 2.1 Crystal Structure of a 1.6-hexanediol Bound Tetrameric Form of Escherichia coli Lac-repressor Refined to 2.1 Resolution
1EFA 2000-03-06 2.6 CRYSTAL STRUCTURE OF THE LAC REPRESSOR DIMER BOUND TO OPERATOR AND THE ANTI-INDUCER ONPF
1TLF 1995-07-31 2.6 UNPRECEDENTED QUATERNARY STRUCTURE OF E. COLI LAC REPRESSOR CORE TETRAMER: IMPLICATIONS FOR DNA LOOPING
1LBI 1996-07-11 2.7 LAC REPRESSOR
4RZS 2015-12-23 2.71 Lac repressor engineered to bind sucralose, unliganded tetramer
1JYF 2001-10-18 3.0 Structure of the Dimeric Lac Repressor with an 11-residue C-terminal Deletion.
4RZT 2015-12-23 3.1 Lac repressor engineered to bind sucralose, sucralose-bound tetramer
1LBH 1996-07-11 3.2 INTACT LACTOSE OPERON REPRESSOR WITH GRATUITOUS INDUCER IPTG
2PAF 2007-06-19 3.5 Crystal Structure of the Lactose Repressor bound to anti-inducer ONPF in induced state
2PE5 2008-03-18 3.5 Crystal Structure of the Lac Repressor bound to ONPG in repressed state
1JWL 2001-10-05 4.0 Structure of the Dimeric lac Repressor/Operator O1/ONPF Complex
1LBG 1996-02-17 4.8 LACTOSE OPERON REPRESSOR BOUND TO 21-BASE PAIR SYMMETRIC OPERATOR DNA, ALPHA CARBONS ONLY

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Lactose operon repressor P03023 LACI_ECOLI