Folding Dynamics of the Trp-Cage Miniprotein: Evidence for a Native-Like Intermediate from Combined Time-Resolved Vibrational Spectroscopy and Molecular Dynamics Simulations

Abstract

Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate. Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530-1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label ((13)C═(18)O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding-unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9-Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

Sampling the equilibrium kinetic network of Trp-cage in explicit solvent

“…In addition, these findings corroborate with the recent IR experiment of Meuzelaar et al 19 Interestingly enough, we found that the intermediate I state, consisting of a formed α-helix with a solvated and 24,28 We can solve this paradox, by constructing the free energy landscape.…”

“…All other timescales are fast processes related to transitions between the metastable states. Recent IR experiments by Meuzelaar et al 19 found biexponential kinetics for the Trp cage system. At a temperature of 300 K, they measured a slow time scale of 2200 ns, and a fast timescale of around 150 ns, which they attribute to the presence of an intermediate.…”

“…Previous measurements of the folding curve yielded p U /p N ≈ 0.33, 15 and more recent IR experiments find a p U /p N ≈ 0.2, close to our predictions. 19 Besides the native N and the unfolded state U, only state SN is significantly populated. The SN state is characterized by a pseudo native structure (Fig.…”

“…This is in line with UV resonance Raman spectroscopy (UVRS) results carried out by Ahmed et al, 18 who proposed the presence of an intermediate with an intact α-helix, where the hydrophobic core is even more compact. Recent T-jump IR experiments 19 established a biexponential relaxation time with τ 1 = 2.2 μs and τ 2 = 150 ns at room temperature which suggested the additional presence of a native-like intermediate, characterized by a partly solvated proline helix.…”

“…14 Notwithstanding its small size, Trpcage contains typical protein motifs, an α-helix (residue 2-8), a 3 10 -helix (residue [11][12][13][14], and a polyproline II helix (residue [17][18][19], which form a hydrophobic core with the tryptophan buried in the center.…”