The origin and impact of bound water around intrinsically disordered proteins.

Proteins and water couple dynamically over a wide range of time scales. Motivated by their central role in protein function, protein-water dynamics and thermodynamics have been extensively studied for structured proteins, where correspondence to structural features has been made. However, properties controlling intrinsically disordered protein (IDP)-water dynamics are not yet known. We report results of megahertz-to-terahertz dielectric spectroscopy and molecular dynamics simulations of a group of IDPs with varying charge content along with structured proteins of similar size. Hydration water around IDPs is found to exhibit more heterogeneous rotational and translational dynamics compared with water around structured proteins of similar size, yielding on average more restricted dynamics around individual residues of IDPs, charged or neutral, compared with structured proteins. The on-average slower water dynamics is found to arise from excess tightly bound water in the first hydration layer, which is related to greater exposure to charged groups. The more tightly bound water to IDPs correlates with the smaller hydration shell found experimentally, and affects entropy associated with protein-water interactions, the contribution of which we estimate based on the dielectric measurements and simulations. Water-IDP dynamic coupling at terahertz frequencies is characterized by the dielectric measurements and simulations.

Change in vibrational entropy with change in protein volume estimated with mode Grüneisen parameters.

For a small adjustment in average volume, due to a change in state of a protein or other macromolecule at constant temperature, the change in vibrational entropy is related to the mode Grüneisen parameters, which relate shifts in frequency to a small volume change. We report here values of mode Grüneisen parameters computed for two hydrated proteins, cytochrome c and myoglobin, which exhibit trends with mode frequency resembling those of glassy systems. We use the mode Grüneisen parameters to relate volumetric thermal expansion to previously computed values of the isothermal compressibility for several proteins. We also estimate changes in vibrational entropy resulting from the change in volume upon ligand bonding of myoglobin and the homodimeric hemoglobin from Scapharca inaequivalvis (HbI). We compare estimates of the change in entropy upon ligation obtained in terms of mode Grüneisen parameters with the results of normal mode analysis for myoglobin and earlier molecular dynamics simulations of HbI. The results illustrate how small changes in average volume can yield changes in entropy that contribute to ligand binding and allostery.

Water-mediated biomolecular dynamics and allostery.

Dynamic coupling with water contributes to regulating the functional dynamics of a biomolecule. We discuss protein-water dynamics, with emphasis on water that is partially confined, and the role of protein-confined water dynamics in allosteric regulation. These properties are illustrated with two systems, a homodimeric hemoglobin from Scapharca inaequivalvis (HbI) and an A2A adenosine receptor (A2AAR). For HbI, water-protein interactions, long known to contribute to the thermodynamics of cooperativity, are seen to influence the dynamics of the protein not only around the protein-water interface but also into the core of each globule, where dynamic and entropic changes upon ligand binding are coupled to protein-water contact dynamics. Similarly, hydration waters trapped deep inside the core region of A2AAR enable the formation of an allosteric network made of water-mediated inter-residue contacts. Extending from the ligand binding pocket to the G-protein binding site, this allosteric network plays key roles in regulating the activity of the receptor.

Hydrophobic Collapse of Ubiquitin Generates Rapid Protein-Water Motions.

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (<500 ns) initial phase of the hydrophobic collapse from the elongated protein to a molten globule structure precedes secondary structure formation. We find that these very first steps, including large-amplitude changes within the unfolded manifold, are accompanied by a rapid (<500 ns) pronounced change of the coupled protein-solvent response. The KITA response upon secondary structure formation exhibits an opposite sign, which indicates a distinct effect on the solvent-exposed surface.

Ensemble characterization of an intrinsically disordered FG-Nup peptide and its F>A mutant in DMSO-d6.

Intrinsically disordered proteins (IDP) lack a well-defined 3D-structure under physiological conditions, yet, the inherent disorder represented by an ensemble of conformation plays a critical role in many cellular and regulatory processes. Nucleoporins, or Nups, are the proteins found in the nuclear pore complex (NPC). The central pore of the NPC is occupied by Nups, which have phenylalanine-glycine domain repeats and are intrinsically disordered, and therefore are termed FG-Nups. These FG-domain repeats exhibit differing cohesiveness character and differ from least (FG) to most (GLFG) cohesive. The designed FG-Nup is a 25 AA model peptide containing a noncohesive FG-motif flanked by two cohesive GLFG-motifs (WT peptide). Complete NMR-based ensemble characterization of this peptide along with a control peptide with an F>A substitution (MU peptide) are discussed. Ensemble characterization of the NMR-determined models suggests that both the peptides do not have consistent secondary structures and continue to be disordered. Nonetheless, the role of cohesive elements mediated by the GLFG motifs is evident in the WT ensemble of structures that are more compact than the MU peptide. The approach presented here allows an alternate way to investigate the specific roles of distinct amino acid motifs that translate into the long-range organization of the ensemble of structures and in general on the nature of IDPs.

Dynamics of Hydrogen Bonds between Water and Intrinsically Disordered and Structured Regions of Proteins.

Recent studies indicate more restricted dynamics of water around intrinsically disordered proteins (IDPs) than structured proteins. We examine here the dynamics of hydrogen bonds between water molecules and two proteins, small ubiquitin-related modifier-1 (SUMO-1) and ubiquitin-conjugating enzyme E2I (UBC9), which we compare around intrinsically disordered regions (IDRs) and structured regions of these proteins. It has been recognized since some time that excluded volume effects, which influence access of water molecules to hydrogen-bonding sites, and the strength of hydrogen bonds between water and protein affect hydrogen bond lifetimes. While we find those two properties to mediate lifetimes of hydrogen bonds between water and protein residues in this study, we also find that the lifetimes are affected by the concentration of charged groups on other nearby residues. These factors are more important in determining the hydrogen bond lifetimes than whether a residue hydrogen bonding with water belongs to an IDR or to a structured region.

Enhanced Mobility during Diels-Alder Reaction: Results of Molecular Simulations.

Recent measurements indicate enhanced mobility of solvent molecules during Diels-Alder (DA) and other common chemical reactions. We present results of molecular dynamics simulations of the last stages of the DA cycloaddition reaction, from the transition state configuration to product, of furfurylamine and maleimide in acetonitrile at reactant concentrations studied experimentally. We find enhanced mobility of solvent and reactant molecules up to at least a nanometer from the DA product over hundreds of picoseconds. Local heating is ruled out as a factor in the enhanced mobility observed in the simulations, which is instead found to be due to solvent relaxation following the formation of the DA product.

Locating and Navigating Energy Transport Networks in Proteins.

We review computational methods to locate energy transport networks in proteins that are based on the calculation of local energy diffusion in nanoscale systems. As an illustrative example, we discuss energy transport networks computed for the homodimeric hemoglobin from Scapharca inaequivalvis, where channels for facile energy transport, which include the cluster of water molecules at the interface of the globules, have been found to lie along pathways that experiments reveal are important in allosteric processes. We also review recent work on master equation simulations to model energy transport dynamics, including efforts to relate rate constants in the master equation to protein structural dynamics. Results for apomyoglobin involving relations between fluctuations in the length of hydrogen bonds and the energy flux between them are presented.

Variation of Energy Transfer Rates across Protein-Water Contacts with Equilibrium Structural Fluctuations of a Homodimeric Hemoglobin.

Molecular dynamics simulations of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) have been carried out to examine relations between rates of vibrational energy transfer across nonbonded contacts and equilibrium structural fluctuations, with emphasis on protein-water contacts. The scaling of rates of energy transfer with equilibrium fluctuations of the contact length is found to hold up well for contacts between residues and hemes at the interface and the cluster of 17 interface water molecules in the unliganded state of HbI, as well as for the liganded state, for which the cluster contains on average 11 water molecules. In both states, the rate of energy transfer is also found to satisfy a diffusion relation. Within each globule, the scaling for polar contacts is similar to that found in an earlier analysis of myoglobin. Entropy associated with dynamics of polar contacts within each globule and with contacts between the hemes and water cluster is found to increase upon ligation. Energy exchange networks (EENs) for liganded and unliganded states obtained from the simulations are also presented and discussed. Energy transport networks through which nonbonded contacts transport energy in HbI, referred to as nonbonded networks (NBNs), are determined from the EENs and compared for the two states.

Energy Transfer across Nonpolar and Polar Contacts in Proteins: Role of Contact Fluctuations.

Molecular dynamics simulations of the villin headpiece subdomain HP36 have been carried out to examine relations between rates of vibrational energy transfer across non-covalently bonded contacts and equilibrium structural fluctuations, with focus on van der Waals contacts. Rates of energy transfer across van der Waals contacts vary inversely with the variance of the contact length, with the same constant of proportionality for all nonpolar contacts of HP36. A similar relation is observed for hydrogen bonds, but the proportionality depends on contact pairs, with hydrogen bonds stabilizing the α-helices all exhibiting the same constant of proportionality, one that is distinct from those computed for other polar contacts. Rates of energy transfer across van der Waals contacts are found to be up to 2 orders of magnitude smaller than rates of energy transfer across polar contacts.

Energy Transport across Interfaces in Biomolecular Systems.

Energy transport during chemical reactions or following photoexcitation in systems of biological molecules is mediated by numerous interfaces that separate chemical groups and molecules. Describing and predicting energy transport has been complicated by the inhomogeneous environment through which it occurs, and general rules are still lacking. We discuss recent work on identification of networks for vibrational energy transport in biomolecules and their environment, with focus on the nature of energy transfer across interfaces. Energy transport is influenced both by structure of the biomolecular system as well as by equilibrium fluctuations of nonbonded contacts between chemical groups, biomolecules, and water along the network. We also discuss recent theoretical and computational work on the related topic of thermal transport through molecular interfaces, with focus on systems important in biology as well as relevant experimental studies.

Scaling of Rates of Vibrational Energy Transfer in Proteins with Equilibrium Dynamics and Entropy.

Theoretical arguments and results of molecular dynamics (MD) simulations of myoglobin at 300 K are presented to relate rates of vibrational energy transfer across nonbonded contacts interacting via short-range potentials to dynamics of the contact. Both theory and the results of the simulations support a scaling relation between the energy transfer rate and the inverse of the variance in the distance between hydrogen-bonded contacts. The results of the MD simulations do not support such a relation for longer-range charged contacts. Instead, the energy transfer rate is found to scale as a power law in the distance between charged groups. The scaling between rates of vibrational energy transfer across nonbonded contacts interacting via short-range potentials and conformational dynamics suggests a relation between vibrational energy transfer rates and entropy associated with the dynamics of interacting residues. The use of time-resolved vibrational spectroscopy to determine change in conformational entropy with change in protein functional state is discussed, and an expression quantifying the connection is provided.

A novel immunofluorescent computed tomography (ICT) method to localise and quantify multiple antigens in large tissue volumes at high resolution.

Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm(3)) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm(3)) at high resolution (<1 µm) and with multiple antigen labelling, for volumetric and quantitative analysis. This is made possible through computer reconstruction of serial sectioned and sequentially immunostained butyl-methyl methacrylate (BMMA) embedded tissue. Using this novel immunofluorescent computed tomography (ICT) approach, we have three-dimensionally reconstructed part of the murine lower eyelid that contains the meibomian gland and localised cell nuclei (DAPI), Ki67 and cytokeratin 1 (CK1), as well as performing non-linear optical (NLO) microscopy imaging of collagen, to assess cell density, cell proliferation, gland keratinisation and gland volume respectively. Antigenicity was maintained after four iterative stains on the same tissue, suggesting that there is no defined limit to the number of antigens that can be immunostained for reconstruction, as long as the sections remain intact and the previous antibody has been successfully eluted. BMMA resin embedding also preserved fluorescence of transgenic proteins. We propose that ICT may provide valuable high resolution, three-dimensional biological maps of multiple biomolecules within a single tissue or organ to better characterise and quantify tissue structure and function.