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

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
2PAF 2007-03-27T00:00:00+0000 3.5 Crystal Structure of the Lactose Repressor bound to anti-inducer ONPF in induced state
1TLF 1995-03-06T00:00:00+0000 2.6 UNPRECEDENTED QUATERNARY STRUCTURE OF E. COLI LAC REPRESSOR CORE TETRAMER: IMPLICATIONS FOR DNA LOOPING
1JWL 2001-09-04T00:00:00+0000 4.0 Structure of the Dimeric lac Repressor/Operator O1/ONPF Complex
1LQC 1996-08-13T00:00:00+0000 0 LAC REPRESSOR HEADPIECE (RESIDUES 1-56), NMR, 32 STRUCTURES
4RZS 2014-12-24T00:00:00+0000 2.71 Lac repressor engineered to bind sucralose, unliganded tetramer
4RZT 2014-12-24T00:00:00+0000 3.1 Lac repressor engineered to bind sucralose, sucralose-bound tetramer
1JYF 2001-09-12T00:00:00+0000 3.0 Structure of the Dimeric Lac Repressor with an 11-residue C-terminal Deletion.
2PE5 2007-04-02T00:00:00+0000 3.5 Crystal Structure of the Lac Repressor bound to ONPG in repressed state
1JYE 2001-09-12T00:00:00+0000 1.7 Structure of a Dimeric Lac Repressor with C-terminal Deletion and K84L Substitution
3EDC 2008-09-03T00:00:00+0000 2.1 Crystal Structure of a 1.6-hexanediol Bound Tetrameric Form of Escherichia coli Lac-repressor Refined to 2.1 Resolution

Relevant UniProtKB Entries

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