Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.


Abstract

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein. Study holds ProTherm entries: 16377, 16378 Extra Details: Experiment conducted at room temperature lactose repressor, coiled-coil structure, lac enzyme

Submission Details

ID: GvMPuzKo

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Barry JK;Matthews KS,Biochemistry (1999) Thermodynamic analysis of unfolding and dissociation in lactose repressor protein. PMID:10350470
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
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

Relevant UniProtKB Entries

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