Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the structure and dynamics of the protein, lysozyme, is investigated by fluorescence correlation spectroscopy (FCS) and molecular dynamic (MD) simulation. The FCS data indicate that addition of the RTIL ([pmim][Br]) leads to reduction in size and faster conformational dynamics of the protein. The hydrodynamic radius (rH) of lysozyme decreases from 18 Å in 0 M [pmim][Br] to 11 Å in 1.5 M [pmim][Br] while the conformational relaxation time decreases from 65 μs to 5 μs. Molecular origin of the collapse (size reduction) of lysozyme in aqueous RTIL is analyzed by MD simulation. The radial distribution function of water, RTIL cation, and RTIL anion from protein clearly indicates that addition of RTIL causes replacement of interfacial water by RTIL cation ([pmim](+)) from the first solvation layer of the protein providing a comparatively dehydrated environment. This preferential solvation of the protein by the RTIL cation extends up to ∼30 Å from the protein surface giving rise to a nanoscopic cage of overall radius 42 Å. In the nanoscopic cage of the RTIL (42 Å), volume fraction of the protein (radius 12 Å) is only about 2%. RTIL anion does not show any preferential solvation near protein surface. Comparison of effective radius obtained from simulation and from FCS data suggests that the "dry" protein (radius 12 Å) alone diffuses in a nanoscopic cage of RTIL (radius 42 Å). MD simulation further reveals a decrease in distance ("domain closure") between the two domains (alpha and beta) of the protein leading to a more compact structure compared to that in the native state.
The hydrophobic effect appears to be a key driving force for many chemical and biological processes, such as protein folding, protein-protein interactions, membrane bilayer self-assembly, and so forth. In this study, we calculated the potential of mean force (PMF) using umbrella sampling technique between different model hydrophobes (methane-methane, cyclobutane-cyclobutane, and between two rodlike hydrophobes) at lower than ambient temperatures (300, 260, and 240 K). We find the appearance of a second solvent-separated minimum at ∼1.0 nm apart from the usual contact and first solvent-separated minimum in the PMF profile of the methane pair at low temperature. In the PMF between both cyclobutane and the rodlike hydrophobe pairs, the second solvent-separated pair (SSSP) becomes even more stable than the first solvent-separated pair (FSSP) at 240 K. Analysis of the water structure shows that, at 240 K, the core water of SSSP for the rodlike hydrophobe pair is more strongly hydrogen bonded and more tetrahedrally oriented than that of the FSSP. Strongly hydrogen-bonded ordered water molecules implicate strong water-water interactions, which are responsible for stabilization of SSSP at low temperature. This weakening of hydrophobic interactions through stabilization of SSSP may play a key role in the cold denaturation of protein.
Despite their routine use as protein denaturants, the comprehensive understanding of the molecular mechanisms by which urea and guanidinium chloride (GdmCl) disrupts proteins' structure is still lacking. Here, we use steered molecular dynamics simulations along with the umbrella sampling technique to elucidate the mechanism of unfolding of chicken villin headpiece (HP-36) in these two denaturants. We find that while urea denatures protein predominantly by forming hydrogen bonds with the protein backbone, GdmCl commences unfolding by weakening of the hydrophobic interactions present in the core. The potential of mean force calculation indicates the reduction of hydrophobic interactions between two benzene moieties in 6 M GdmCl as compared to 6 M urea. We observe a near parallel orientation between the guanidinium cation and aromatic side chains of the HP-36 suggesting π-cation type stacking interactions which play a crucial role in weakening of the hydrophobic interaction. We use QM/MM optimization calculations to estimate the energetics of this π-cation interaction. Additionally, the consistency of the unfolding paths between high temperature (400 K) unfolding simulations and steered molecular dynamics simulations strengthens the proposed molecular mechanism of unfolding further.
The primary driving force for protein folding is the formation of a well-packed, anhydrous core. However, recently, the crystal structure of an antifreeze protein, maxi, has been resolved where the core of the protein is filled with water, which apparently contradicts the existing notion of protein folding. Here, we have performed standard molecular dynamics (MD) simulation, replica exchange MD (REMD) simulation, and umbrella sampling using TIP4P water at various temperatures (300, 260, and 240 K) to explore the origin of this unusual structural feature. It is evident from standard MD and REMD simulations that the protein is found to be stable at 240 K in its unusual state. The core of protein has two layers of semi-clathrate water separating the methyl groups of alanine residues from different helical strands. However, with increasing temperature (260 and 300 K), the stability decreases as the core becomes dehydrated, and methyl groups of alanine are tightly packed driven by hydrophobic interactions. Calculation of the potential of mean force by an umbrella sampling technique between a pair of model hydrophobes resembling maxi protein at 240 K shows the stabilization of second solvent-separated minima (SSM), which provides a thermodynamic rationale of the unusual structural feature in terms of weakening of the hydrophobic interaction. Because the stabilization of SSMs is implicated for cold denaturation, it suggests that the maxi protein is so designed by nature where the cold denaturedlike state becomes the biologically active form as it works near or below the freezing point of water.
Clathrate hydrate forms when a hydrophobic molecule is entrapped inside a water cage or cavity. Although biomolecular structures also have hydrophobic patches, clathrate-like water is found in only a limited number of biomolecules. Also, while clathrate hydrates form at low temperature and moderately higher pressure, clathrate-like water is observed in biomolecular structure at ambient temperature and pressure. These indicate presence of other factors along with hydrophobic environment behind the formation of clathrate-like water in biomolecules. In the current study, we presented a systematic approach to explore the factors behind the formation of clathrate-like water in biomolecules by means of molecular dynamics simulation of a model protein, maxi, which is a naturally occurring nanopore and has clathrate-like water inside the pore. Removal of either confinement or hydrophobic environment results in the disappearance of clathrate-like water ordering, indicating a coupled role of these two factors. Apart from these two factors, clathrate-like water ordering also requires anchoring groups that can stabilize the clathrate-like water through hydrogen bonding. Our results uncover crucial factors for the stabilization of clathrate-like ordering in biomolecular structure which can be used for the development of new biomolecular structure promoting clathrate formation.
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