Site-Specific Coupling of Hydration Water and Protein Flexibility Studied in Solution with Ultrafast 2D-IR Spectroscopy

Abstract

There is considerable evidence for the slaving of biomolecular dynamics to the motions of the surrounding solvent environment, but to date there have been few direct experimental measurements capable of site-selectively probing both the dynamics of the water and the protein with ultrafast time resolution. Here, two-dimensional infrared spectroscopy (2D-IR) is used to study the ultrafast hydration and protein dynamics sensed by a metal carbonyl vibrational probe covalently attached to the surface of hen egg white lysozyme dissolved in D(2)O/glycerol solutions. Surface labeling provides direct access to the dynamics at the protein-water interface, where both the hydration and the protein dynamics can be observed simultaneously through the vibrational probe's frequency-frequency correlation function. In pure D(2)O, the correlation function shows a fast initial 3 ps decay corresponding to fluctuations of the hydration water, followed by a significant static offset attributed to fluctuations of the protein that are not sampled within the <20 ps experimental window. Adding glycerol increases the bulk solvent viscosity while leaving the protein structurally intact and hydrated. The hydration dynamics exhibit a greater than 3-fold slowdown between 0 and 80% glycerol (v/v), and the contribution from the protein's dynamics is found to slow in a nearly identical fashion. In addition, the magnitude of the dynamic slowdown associated with hydrophobic hydration is directly measured and shows quantitative agreement with predictions from molecular dynamics simulations.

Effect of viscogens on the kinetic response of a photoperturbed allosteric protein

“…63,64 This conclusion has found support from experimental techniques such as high precision densimetry, 46 neutron scattering experiments, 47 or the vibrational lifetime of a surface exposed protein labels. 48 While the question whether or not specific interactions of the viscogen molecule with the protein play a role might depend on the particular process studied, in our case they clearly do. In this regard it might be worth mentioning that the structural change initiated by the photoswitch is very small with an RMSD of 0.8-0.9 Å according to NMR structure analysis.…”

“…For example, it has been argued that both glycerol and sucrose are highly preferably excluded from the protein surface, [46][47][48] that gylcerol preferentially hydrates proteins, 60 and that these viscogens affects ligand dynamics in myoglobin only via the solvent viscosity, but not in a viscogen-specific way. 3 On the other hand, these viscogens also act as osmolytes and/or crowders, which often work through direct interactions with the protein, e.g., by attenuating the H-bond strength between polar protein residues and water, 61 or by interaction to the protein backbone with different strength.…”

“…To reach that value, a concentration of 40% w/w was needed for sucrose and 54% w/w for glycerol (see glycerol: MW 92), but both have been argued to be highly preferably excluded from the protein surface. [46][47][48] Nevertheless, Fig. 7 shows that glycerol significantly slows the relaxation kinetics in comparison with sucrose.…”

Solvation of fluoro-acetonitrile in water by 2D-IR spectroscopy: A combined experimental-computational study

“…10) or the CN − (Refs. 11 and 12) ion in water, the -C= =O group of N-methylacetamide (NMA) in water, 13,14 the phosphate group of hydrated phospholipids, 15 the OH/OD vibration of HOD in either D 2 O or H 2 O, [16][17][18][19] -C≡ ≡O, -C≡ ≡N, -N − 3 or -SCN ligands, [20][21][22][23][24] or as unnatural amino acids [25][26][27][28] inside proteins, to name just a few.…”

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

“…In fact the original purpose for which amino acids containing an azide group has been designed was "click" chemistry, and consequently one may also add metal-carbonyl complexes with even stronger C≡O bands to it in a post-translational coupling step as a second alternative [48]. Finally, Re-carbonyl or Ru-carbonyl complexes have been bound selectively to histidines [30,[56][57][58].…”