The experimental folding landscape of monomeric lactose repressor, a large two-domain protein, involves two kinetic intermediates.
To probe the experimental folding behavior of a large protein with complex topology, we created a monomeric variant of the lactose repressor protein (MLAc), a well characterized tetrameric protein that regulates transcription of the lac operon. Purified MLAc is folded, fully functional, and binds the inducer isopropyl beta-d-thiogalactoside with the same affinity as wild-type LacI. Equilibrium unfolding of MLAc induced by the chemical denaturant urea is a reversible, apparent two-state process (pH 7.5, 20 degrees C). However, time-resolved experiments demonstrate that unfolding is single-exponential, whereas refolding data indicate two transient intermediates. The data reveal the initial formation of a burst-phase (tau < ms) intermediate that corresponds to approximately 50% of the total secondary-structure content. This step is followed by a rearrangement reaction that is rate-limited by an unfolding process (tau approximately 3 s; pH 7.5, 20 degrees C) and results in a second intermediate. This MLAc intermediate converts to the native structure (tau approximately 30 s; pH 7.5, 20 degrees C). Remarkably, the experimental folding-energy landscape for MLAc is in excellent agreement with theoretical predictions using a simple topology-based C(alpha)-model as presented in a companion article in this issue.
Wilson
Corey J
CJ
Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, and Department of Chemistry, Rice University, 6100 Main Street, Houston, TX 77251, USA.
Das
Payel
P
Clementi
Cecilia
C
Matthews
Kathleen S
KS
Wittung-Stafshede
Pernilla
P
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D055592:Biophysical Phenomena; D001703:Biophysics; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D056968:Lac Repressors; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins