Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy

Abstract

A series of two-dimensional infrared vibrational echo experiments performed on nitrile-labeled villin headpiece [HP35-ðCNÞ 2 ] is described. HP35 is a small peptide composed of three alpha helices in the folded configuration. The dynamics of the folded HP35-ðCNÞ 2 are compared to that of the guanidine-induced unfolded peptide, as well as the nitrile-functionalized phenylalanine (PheCN), which is used to differentiate the peptide dynamic contributions to the observables from those of the water solvent. Because the viscosity of solvent has a significant effect on fast dynamics, the viscosity of the solvent is held constant by adding glycerol. For the folded peptide, the addition of glycerol to the water solvent causes observable slowing of the peptide's dynamics. Holding the viscosity constant as GuHCl is added, the dynamics of unfolded peptide are much faster than those of the folded peptide, and they are very similar to that of PheCN. These observations indicate that the local environment of the nitrile in the unfolded peptide resembles that of PheCN, and the dynamics probed by the CN are dominated by the fluctuations of the solvent molecules, in contrast to the observations on the folded peptide.nitrile IR probe | peptide dynamics T he structure of proteins, both folded and unfolded, are inherently dynamic in nature. A protein is constantly undergoing interconversions among a range of structures separated by relatively low energy barriers. Fast conformational sampling can give rise to protein structural evolution on slower time scales. Many experimental and theoretical studies have focused on the native structure due to the relevance of the native state protein to its biological functions, but also because of difficulties associated with characterizing proteins under unfolding conditions, where they can exist in heterogeneous distributions of many conformations (1, 2). Properties of the unfolded state are also significant because they play important roles in folding and stability, transport across membranes, and proteolysis and protein turnover (1). Unfolded proteins have dynamics that are different from the native protein, and the differences in dynamics can shed light on the relationship between the native and the unfolded protein.An attractive target for studying differences between the folded and unfolded structures is the villin headpiece 35 (HP35), a peptide chain composed of 35 amino acids found in a chicken villin as a small subdomain. HP35 folds fast (∼1 μs) compared to large proteins, allowing tractable folding/unfolding simulations. Consequently, it has served as a model system in a number of computational folding studies (3-5), as well as a variety of experimental studies (6-8). NMR and X-ray diffraction studies have indicated that wild-type HP35 folds into three alpha helices (Fig. 1), which encase the hydrophobic core composed of three phenylalanines (9, 10).In this paper, the results of a series of ultrafast 2D IR vibrational echo experiments on nitrile-incorporated HP35 are presented. Two CN-fun...

Microscopic insights into the protein-stabilizing effect of trimethylamine N-oxide (TMAO)

“…most likely reports on the dynamics of H-bond formation and breaking between the nitrile group and water, which cause the vibrational frequencies to fluctuate (49-51). As shown (Table 2), the decay time constant of this Kubo term determined for GF CN G in water is about 2.2 ps, which is in good agreement with previously reported values for the nitrile stretching vibration in aqueous solutions (30,37,46,52). In addition, the offset term B obtained in water is practically zero.…”

“…2 and the resultant fitting parameters (i.e., T 2 *, Δ s , and Δ) are listed in Table 1. In particular, the parameters obtained for water are in good agreement with those previously reported for this probe (37). With the knowledge of T 2 *, we further calculated the pure dephasing linewidth (Γ) for each case using the relationship of Γ = 1/πT 2 *.…”

“…Because several studies have suggested that protecting osmolytes, such as TMAO, could directly interact with aromatic sidechains (14), the second mutant we chose to study contains a Phe58/Phe CN mutation at the core of the protein (referred to as HP35-P58). Because the Phe CN residue in HP35-P58 is, to a large extent, buried in a hydrophobic environment (37,53,61), it is unlikely that TMAO will show any direct interactions with the nitrile probe. Thus, results obtained from both mutants will allow us to gain a more complete assessment of the effect of TMAO on protein stability.…”

“…For an IR probe that is able to interact with water via H bonding, measurement of its FFCF can provide, sometimes in a site-specific manner, detailed information about the hydration dynamics of the protein molecule of interest. For example, this approach has been used to identify the existence of mobile water molecules inside Aβ40 amyloid fibrils (31,32) and to interrogate the water-assisted drug-binding mechanism of HIV-1 reverse transcriptase (33), among many other applications (34)(35)(36)(37)(38)(39). In the current study, we capitalize on the established sensitivity of the nitrile stretching vibration (C≡N) to local hydration and electrostatic environment (40) and use the unnatural amino acid p-cyano-phenyalanine (Phe CN ) as a local IR reporter.…”

Transient 2D-IR spectroscopy of inorganic excited states

“…Having said this, 2D-IR offers a relatively direct, graphical approach to extracting the data and so has become a popular approach, being employed for biologically-related samples as well as solute-solvent systems. 11,26,[64][65][66][67][68][69][70] Transferring this experiment to the excited state has been achieved. The first instance of this was the application of T-2D-IR spectroscopy to observe the behaviour of the symmetric CO stretching vibration of Re(CO) 3 Cl(dmbpy) upon excitation of the metal to ligand charge transfer transition in DMSO.…”