The experimental folding landscape of monomeric lactose repressor, a large two-domain protein, involves two kinetic intermediates

Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14563-8. doi: 10.1073/pnas.0505808102. Epub 2005 Oct 3.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / isolation & purification*
  • Biophysical Phenomena
  • Biophysics
  • Escherichia coli
  • Escherichia coli Proteins
  • Kinetics
  • Lac Repressors
  • Models, Molecular*
  • Protein Binding
  • Protein Folding*
  • Repressor Proteins / chemistry*
  • Repressor Proteins / isolation & purification*

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Lac Repressors
  • LacI protein, E coli
  • Repressor Proteins