How Quickly Can a β-Hairpin Fold from Its Transition State?

Abstract

Understanding the structural nature of the free energy bottleneck(s) encountered in protein folding is essential to elucidating the underlying dynamics and mechanism. For this reason, several techniques, including Φ-value analysis, have previously been developed to infer the structural characteristics of such high free-energy or transition states. Herein we propose that one (or few) appropriately placed backbone and/or side chain cross-linkers, such as disulfides, could be used to populate a thermodynamically accessible conformational state that mimics the folding transition state. Specifically, we test this hypothesis on a model β-hairpin, Trpzip4, as its folding mechanism has been extensively studied and is well understood. Our results show that cross-linking the two β-strands near the turn region increases the folding rate by an order of magnitude, to about (500 ns)−1, whereas cross-linking the termini results in a hyperstable β-hairpin that has essentially the same folding rate as the uncross-linked peptide. Taken together, these findings suggest that cross-linking is not only a useful strategy to manipulate folding free energy barriers, as shown in other studies, but also, in some cases, it can be used to stabilize a folding transition state analogue and allow for direct assessment of the folding process on the downhill side of the free energy barrier. The calculated free energy landscape of the cross-linked Trpzip4 also supports this picture. An empirical analysis further suggests, when folding of β-hairpins does not involve a significant free energy barrier, the folding time (τ) follows a power law dependence on the number of hydrogen bonds to be formed (nH), namely, τ = τ0nHα, with τ0 = 20 ns and α = 2.3.

Biomolecular Crowding Arising from Small Molecules, Molecular Constraints, Surface Packing, and Nano-Confinement

“…In addition, as highlighted above, a specifically engineered crowded environment can be created, for example, by increasing the local mass density or through confinement and the resultant crowding effect can be investigated to provide insight into various biochemical and biophysical questions. In particular, we believe that the strategy of using a structural motif to crowd or constrain a specific volume element in the biomolecule of interest has the potential to be useful in the following applications: (1) to help assess the magnitude of internal friction encountered in protein folding dynamics, (2) to trap a high free energy state, 78,79 and (3) to modulate the on- and off-rates of ligand binding by exposing binding sites in proteins using a photolyzable constraint. Similarly, the confining strategy via RM inclusion could be extended to, for example, (1) study the excluded volume effect on the structure and dynamics of intrinsically disordered proteins or other metastable proteins, 80 (2) study the structure and folding dynamics of peptides and proteins that develop secondary and/or tertiary interactions at a low degree of hydration such as the late embryogenesis abundant (LEA) proteins, which fold into α-helices upon desiccation, 81 (3) expand the time window of observation in laser-induced temperature-jump experiments 82 using an organic solvent (e.g, isooctane) that has a smaller thermal conductivity than water to slow down the rate of heat transfer from the water pool in the RMs to the surroundings, and (4) investigate the water molecules inside of the cytosol of E. coli cells using infrared spectroscopy by removing bulk water contributions through RM encapsulation.…”

Effect of heterochiral inversions on the structure of a β‐hairpin peptide

“…The 16‐residue long GB1 peptide, the C‐terminal hairpin (residues 41‐56) of streptococcal Protein G's B1 domain, is a representative of a large family of peptides featuring the β‐hairpin structural motif. The folding mechanism of β‐hairpins has been studied in the literature, both experimentally and via simulations . Two key events have been identified, namely formation of a stable turn and formation of cross‐strand hydrogen bonds and hydrophobic contacts.…”

Infrared and Fluorescence Assessment of Protein Dynamics: From Folding to Function

“…In a proof-of-principle study, 87 we showed that it is possible, at least for simple protein systems, to engineer TS analogs for assessing these type of dynamics. Using a tryptophan zipper, 88 Trpzip4, as a model, we aimed to stabilize a specific native contact formed in the TS using a suitable covalent bond.…”