Folding Dynamics and Pathways of the Trp-Cage Miniproteins

Abstract

Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9–7.5 μs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.

FF12MC: A revised AMBER forcefield and new protein simulation protocol

“…1(O)]. More importantly, the τ f s of 1998 ns smt at 280 K for the Trp‐cage (TC10b)—obtained by using the Kaplan‐Meier estimator without any prior knowledge of the hazard function for the nonnative state population of the miniprotein—is consistent with the experimentally determined τ f of 1430 ns smt at 300 K . Plotting the natural logarithm of the nonnative state population versus time‐to‐folding from nonnative states to the native state of the Trp‐cage reveals a linear relationship with r 2 of 0.9408 (Fig.…”

“…Without any prior knowledge of the hazard function for the nonnative state population of the Trp‐cage, the τ f of the miniprotein was predicted to be 1998 ns smt (95% CI: 1396 – 2860 ns smt ) from the 30 simulations at 280 K (Table ). This τ f is consistent with the experimentally determined τ f of 1430 ns smt at 300 K . By comparison, the folding time of the same Trp‐cage sequence reported to date is 14 μs smt that was estimated—also without any prior knowledge that the Trp‐cage follows a two‐state kinetics scheme—from a pioneering 208‐μs smt canonical MD simulation performed on a one‐of‐a‐kind extremely powerful special‐purpose supercomputer .…”

“…), indicating an exponential decay of the nonnative state population of the Trp‐cage over simulation time. This exponential decay is in excellent agreement with the experimental observation that the folding of Trp‐cage follows a two‐state kinetics scheme . These results show that FF12MC can fold the Trp‐cage from scratch with high accuracy in the 30 simulations at 280 K. Further, the results demonstrate that FF12MC can capture the two‐state kinetics scheme as the major folding pathway of the Trp‐cage with an estimated τ f that is consistent with the experimental value.…”

“…This article reports an FF12MC evaluation study consisting of 1350 NPT MD simulations at 1 atm and 274–340 K with an aggregated simulation time of 1252.572 μs smt . Using general‐purpose AMBER forcefields FF96 (see RESULTS AND DISCUSSION for reasons to include this forcefield), FF12SB, and FF14SB as references, these simulations were carried out to determine whether in multiple, distinct, independent, unrestricted, unbiased, and classical NPT MD simulations FF12MC or FF12MCsm can (i) reproduce the experimental J ‐coupling constants of four cationic homopeptides (Ala 3 , Ala 5 , Ala 7 , and Val 3 ) and four folded globular proteins of the third immunoglobulin‐binding domain of protein G (GB3), bovine pancreatic trypsin inhibitor (BPTI), ubiquitin, and lysozme, (ii) reproduce crystallographic B‐factors and nuclear magnetic resonance (NMR)‐derived Lipari‐Szabo order parameters of GB3, BPTI, ubiquitin, and lysozyme, (iii) simulate the experimentally observed flipping between left‐ and right‐handed configurations for the C14–C38 disulfide bond of BPTI and its mutant, (iv) autonomously fold β‐hairpins of chignolin and CLN025 and an α‐miniprotein Trp‐cage (the TC10b sequence) with folding times (τ f s) in agreement with experimental τ f s, (v) simulate subsequent unfolding and refolding of these sequences, and (vi) refine TMR01, TMR04, and TMR07—comparative models of proteins selected from the first Critical Assessment of protein Structure Prediction model Refinement (CASPR) experiment (http://predictioncenter.org/caspR/, note that subsequent model refinement experiments are called CASP rather than CASPR). Unless otherwise specified, all simulations described below are multiple, distinct, independent, unrestricted, unbiased, and classical NPT MD simulations.…”

Examination of ethanol interactions with Trp‐cage peptide through MD simulations and intermolecular nuclear Overhauser effects

“…The “caged” tryptophan6 residue is surrounded by hydrophobic side chains, including those of Tyr3, Leu7, Pro12, Pro18, and Pro 19. Trp‐cage is small enough that studies of its structure and conformational dynamics by a variety of experiments and computational approaches are practical. …”