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

AUTOR(ES)

Wilson, Corey J.

FONTE

National Academy of Sciences

RESUMO

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 β-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°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 (τ < ms) intermediate that corresponds to ≈50% of the total secondary-structure content. This step is followed by a rearrangement reaction that is rate-limited by an unfolding process (τ ≈ 3 s; pH 7.5, 20°C) and results in a second intermediate. This MLAc intermediate converts to the native structure (τ ≈ 30 s; pH 7.5, 20°C). Remarkably, the experimental folding-energy landscape for MLAc is in excellent agreement with theoretical predictions using a simple topology-based Cα-model as presented in a companion article in this issue.

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