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
ID: aoDXnWZ6
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
Version: 1
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Structure ID | Release Date | Resolution | Structure Title |
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1CJG | 1999-04-14T00:00:00+0000 | 0 | NMR STRUCTURE OF LAC REPRESSOR HP62-DNA COMPLEX |
1EFA | 2000-02-07T00:00:00+0000 | 2.6 | CRYSTAL STRUCTURE OF THE LAC REPRESSOR DIMER BOUND TO OPERATOR AND THE ANTI-INDUCER ONPF |
1JWL | 2001-09-04T00:00:00+0000 | 4.0 | Structure of the Dimeric lac Repressor/Operator O1/ONPF Complex |
1JYE | 2001-09-12T00:00:00+0000 | 1.7 | Structure of a Dimeric Lac Repressor with C-terminal Deletion and K84L Substitution |
1JYF | 2001-09-12T00:00:00+0000 | 3.0 | Structure of the Dimeric Lac Repressor with an 11-residue C-terminal Deletion. |
1L1M | 2002-02-19T00:00:00+0000 | 0 | SOLUTION STRUCTURE OF A DIMER OF LAC REPRESSOR DNA-BINDING DOMAIN COMPLEXED TO ITS NATURAL OPERATOR O1 |
1LBG | 1996-01-03T00:00:00+0000 | 4.8 | LACTOSE OPERON REPRESSOR BOUND TO 21-BASE PAIR SYMMETRIC OPERATOR DNA, ALPHA CARBONS ONLY |
1LBH | 1996-02-17T00:00:00+0000 | 3.2 | INTACT LACTOSE OPERON REPRESSOR WITH GRATUITOUS INDUCER IPTG |
1LBI | 1996-02-17T00:00:00+0000 | 2.7 | LAC REPRESSOR |
1LCC | 1993-03-25T00:00:00+0000 | 0 | 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 |
Percent Identity | Matching Chains | Protein | Accession | Entry Name |
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100.0 | Lactose operon repressor | P03023 | LACI_ECOLI |