A detailed unfolding pathway of a (β/α)₈‐barrel protein as studied by molecular dynamics simulations

Abstract

The (beta/alpha)(8)-barrel is the most common protein fold. Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two beta-strands and three alpha-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation. To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli were carried out using a special-purpose parallel computer system. Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events. Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies. Then, denaturation of the first two betaalpha units and separation of the sixth strand from the fifth took place independently. The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold. Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module.

Identification and Characterization of Key Substructures Involved in the Early Folding Events of a (β/α)8-barrel Protein as Studied by Experimental and Computational Methods

“…The results of our previous ePRAI unfolding simulations suggest that separation of b 6 and b 5 is unlikely to affect the subsequent unfolding of (b/a) 1-4 b 5 . 24 Since removal of secondary structure elements from either the N or C terminus of (b/a) 1-4 b 5 seems to prevent folding altogether, (b/a) 1-4 b 5 is the smallest segment that is capable of autonomous folding. Covalent addition of (b/a) 6 to (b/a) 1-4 b 5 , yielding (b/a) 1-6 , causes a significant increase in conformational stability.…”

“…24 Here, the unfolding simulations were continued to 20 ns and 12 ns at 500 K and 525 K, respectively, to produce an ensemble of unfolded structures. We sampled structures present between 18 ns and 20 ns at 500 K and structures present between 10 ns and 12 ns at 525 K. These structures represent the denatured state.…”

“…An ensemble of unfolding intermediates, with structural properties similar to the equilibrium folding intermediates, appeared during our previous molecular dynamics (MD) simulation. 24 In that study, we computed MD trajectories for temperature-induced ePRAI unfolding so that its entire folding/unfolding pathway could be characterized. These intermediates probably appear relatively late in folding.…”

Identifying critical residues in protein folding: Insights from ϕ-value and Pfold analysis

“…Interestingly, the very first microscopic evidence for the existence of a nucleation mechanism in protein folding was obtained in the scope of Monte Carlo ͑MC͒ simulations of a simple lattice model, where Abkevich et al 9 observed that once the FN, consisting of a specific set of native bonds, is established the native fold is achieved very rapidly. Additional studies in vitro [10][11][12][13] and in silico, [14][15][16][17][18][19][20][21][22][23][24] using more sophisticated protein models and other simulational methodologies, have provided further evidence for the existence of nucleation sites in CI2 as well as in other target proteins. For this reason the nucleation mechanism is typically considered the most common folding mechanism among small two-state proteins.…”

Experimental Evidence for the Existence of a Stable Half-Barrel Subdomain in the (β/α)8-Barrel Fold

“…Thus, (β/α) 3-6 is an independent folding unit and can serve as an incipient folding scaffold that may guide the remainder of the polypeptide chain to the correct folding pathway, as predicted by a previous computer simulation study. 28 …”