The Folding Energy Landscape of the Dimerization Domain of Escherichia coli Trp Repressor: A Joint Experimental and Theoretical Investigation

Abstract

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of E. coli tryptophan repressor, 2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66] 2 TR are sufficient to drive the formation of a stable dimer for the fulllength protein, [2-107] 2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66] 2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66] 2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and MALDI mass spectrometry performed at several protein and denaturant concentrations. Peptides corresponding to all three helices in [2-66] 2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the A-helix and another from the C-helix may reflect non-random structure, possibly a pre-cursor for segment swapping, in the urea-denatured state. Native topologybased model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX-MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.

A versatile method for systematic conformational searches: Application to CheY

“…The energy vs. RMSD distributions tended to be funnel-shaped, with large numbers of structures at higher energies and fewer structures, usually closer to native, at lower energies, similar to previous observations for protein folding landscapes [37, 60, 61, 62, 63, 64]. In the reworking stage, the native structure was found to reside in a deep, narrow minimum on the energy surface, a finding consistent with prior studies of CheY[37].…”

Nonnative Electrostatic Interactions Can Modulate Protein Folding: Molecular Dynamics with a Grain of Salt

“…This assumption describes folding as occurring on a perfectly funneled landscape, and its relevance was proven by various studies that use native-topology-based models. [4][5][6][7][8][9][10][11][12][13] While various aspects of the complexity of protein folding are correctly addressed by assuming a perfectly funneled landscape, it is clear that protein sequences are frustrated to some extent. Yet, the exact magnitude of frustration in proteins is rather unclear.…”

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…This approach has previously been used to determine the relative percentage protection of peptides after HD exchange. 23,24,62 …”