RESUMEN

O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Asunto(s)

RESUMEN

Confinement of matter on the nanometre scale can induce phase transitions not seen in bulk systems. In the case of water, so-called drying transitions occur on this scale as a result of strong hydrogen-bonding between water molecules, which can cause the liquid to recede from nonpolar surfaces to form a vapour layer separating the bulk phase from the surface. Here we report molecular dynamics simulations showing spontaneous and continuous filling of a nonpolar carbon nanotube with a one-dimensionally ordered chain of water molecules. Although the molecules forming the chain are in chemical and thermal equilibrium with the surrounding bath, we observe pulse-like transmission of water through the nanotube. These transmission bursts result from the tight hydrogen-bonding network inside the tube, which ensures that density fluctuations in the surrounding bath lead to concerted and rapid motion along the tube axis. We also find that a minute reduction in the attraction between the tube wall and water dramatically affects pore hydration, leading to sharp, two-state transitions between empty and filled states on a nanosecond timescale. These observations suggest that carbon nanotubes, with their rigid nonpolar structures, might be exploited as unique molecular channels for water and protons, with the channel occupancy and conductivity tunable by changes in the local channel polarity and solvent conditions.

Asunto(s)

RESUMEN

Laser tweezers and atomic force microscopes are increasingly used to probe the interactions and mechanical properties of individual molecules. Unfortunately, using such time-dependent perturbations to force rare molecular events also drives the system away from equilibrium. Nevertheless, we show how equilibrium free energy profiles can be extracted rigorously from repeated nonequilibrium force measurements on the basis of an extension of Jarzynski's remarkable identity between free energies and the irreversible work.

RESUMEN

We study the reversible folding/unfolding of short Ala and Gly-based peptides by molecular dynamics simulations of all-atom models in explicit water solvent. A kinetic analysis shows that the formation of a first alpha-helical turn occurs within 0.1-1 ns, in agreement with the analyses of laser temperature jump experiments. The unfolding times exhibit Arrhenius temperature dependence. For a rapidly nucleating all-Ala peptide, the helix nucleation time depends only weakly on temperature. For a peptide with enthalpically competing turn-like structures, helix nucleation exhibits an Arrhenius temperature dependence, corresponding to the unfolding of enthalpic traps in the coil ensemble. An analysis of structures in a "transition-state ensemble" shows that helix-to-coil transitions occur predominantly through breaking of hydrogen bonds at the helix ends, particularly at the C-terminus. The temperature dependence of the transition-state ensemble and the corresponding folding/unfolding pathways illustrate that folding mechanisms can change with temperature, possibly complicating the interpretation of high-temperature unfolding simulations. The timescale of helix formation is an essential factor in molecular models of protein folding. The rapid helix nucleation observed here suggests that transient helices form early in the folding event.

Asunto(s)

RESUMEN

The time, temperature, and sequence dependences of helix formation kinetics of fully atomistic peptide models in explicit solvent are described quantitatively by a diffusive search within the coil state with barrierless transitions into the helical state. Conformational diffusion leads to nonexponential kinetics and jump-width dependences in temperature jump experiments.

Asunto(s)

RESUMEN

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.

Asunto(s)

RESUMEN

Surgery for the repair of a type I aortic dissection presents several difficulties for the surgeon and the perfusionist. One must safely support the patient, while at the same time provide the surgeon with a bloodless field in which to operate. Often, this requires cessation of the circulation for varying amounts of time. Deep hypothermia allows for an extension of the arrest period, while other techniques-- retrograde cerebral perfusion and antegrade cerebral perfusion--provide an additional degree of cerebral protection. Recently, we utilized these techniques concurrently on a 43-year-old female who presented for a reoperation for a type I aortic dissection. Combining these techniques allowed us to adequately support the patient during an anticipated lengthy period of circulatory arrest and insured a successful operation without any adverse cerebral or other organ dysfunction.

Asunto(s)

RESUMEN

The kinetics of water penetration and escape in cytochrome c (cyt c) is studied by molecular dynamics (MD) simulations at various temperatures. Water molecules that penetrate the protein interior during the course of an MD simulation are identified by monitoring the number of water molecules in the first coordination shell (within 3.5 A) of each water molecule in the system. Water molecules in the interior of cyt c have 0-3 water molecules in their first hydration shell and this coordination number persists for extended periods of time. At T = 300 K we identify over 200 events in which water molecules penetrate the protein and reside inside for at least 5 picoseconds (ps) within a 1.5 nanoseconds (ns) time period. Twenty-seven (27) water molecules reside for at least 300 ps, 17 water molecules reside in the protein interior for times longer than 500 ps, and two interior water molecules do not escape; at T = 360 K one water molecule does not escape; at 430 K all water molecules exchange. Some of the internal water molecules show mean square displacements (MSD) of 1 A2 characteristic of structural waters. Others show MSD as large as 12 A2, suggesting that some of these water molecules occupy transient cavities and diffuse extensively within the protein. Motions of protein-bound water molecules are rotationally hindred, but show large librations. Analysis of the kinetics of water escape in terms of a survival time correlation function shows a power law behavior in time that can be interpreted in terms of a broad distribution of energy barriers, relative to kappa BT, for water exchange. At T = 300 K estimates of the roughness of the activation energy distribution is 4-10 kJ/mol (2-4 kappa BT). Activation enthalpies for water escape are 6-23 kJ/mol. The difference in activation entropies between fast exchanging (0.01 ns) and slow exchanging (0.1-1 ns) water molecules is -27 J/K/mol. Dunitz (Science 1997;264:670.) has estimated the maximum entropy loss of a water molecule due to binding to be 28 J/K/mol. Therefore, our results suggest that the entropy of interior water molecules is similar to entropy of bulk water.

Asunto(s)

RESUMEN

We study the dynamical fluctuations of horse heart cytochrome c by molecular dynamics (MD) simulations in aqueous solution, at four temperatures: 300 K, 360 K, 430 K, and 550 K. Each simulation covers a production time of at least 1.5 nanoseconds (ns). The conformational dynamics of the system is analyzed in terms of collective motions that involve the whole protein, and local motions that involve the formation and breaking of intramolecular hydrogen bonds. The character of the MD trajectories can be described within the framework of rugged energy landscape dynamics. The MD trajectories sample multiple conformational minima, with basins in protein conformational space being sampled for a few hundred picoseconds. The trajectories of the system in configurational space can be described in terms of diffusion of a particle in real space with a waiting time distribution due to partial trapping in shallow minima. As a consequence of the hierarchical nature of the dynamics, the mean square displacement autocorrelation function, <|x(t) - x(0)|2>, exhibits a power law dependence on time, with an exponent of around 0.5 for times shorter than 100 ps, and an exponent of 1.75 for longer times. This power law behavior indicates that the system exhibits suppressed diffusion (sub-diffusion) in sampling of configurational space at time scales shorter than 100 ps, and enhanced (super-diffusion) at longer time scales. The multi-basin feature of the trajectories is present at all temperatures simulated. Structural changes associated with inter-basin displacements correspond to collective motions of the Omega loops and coiled regions and relative motions of the alpha-helices as rigid bodies. Similar motions may be involved in experimentally observed amide hydrogen exchange. However, some groups showing large correlated motions do not expose the amino hydrogens to the solvent. We show that large fluctuations are not necessarily correlated to hydrogen exchange. For example, regions of the proteins forming alpha helices and turns show significant fluctuations, but as rigid bodies, and the hydrogen bonds involved in the formation of these structures do not break in proportion to these fluctuations. Proteins 1999;36:175-191. Published 1999 Wiley-Liss, Inc.

Asunto(s)

RESUMEN

Conformational free energies of butane, pentane, and hexane in water are calculated from molecular simulations with explicit waters and from a simple molecular theory in which the local hydration structure is estimated based on a proximity approximation. This proximity approximation uses only the two nearest carbon atoms on the alkane to predict the local water density at a given point in space. Conformational free energies of hydration are subsequently calculated using a free energy perturbation method. Quantitative agreement is found between the free energies obtained from simulations and theory. Moreover, free energy calculations using this proximity approximation are approximately four orders of magnitude faster than those based on explicit water simulations. Our results demonstrate the accuracy and utility of the proximity approximation for predicting water structure as the basis for a quantitative description of n-alkane conformational equilibria in water. In addition, the proximity approximation provides a molecular foundation for extending predictions of water structure and hydration thermodynamic properties of simple hydrophobic solutes to larger clusters or assemblies of hydrophobic solutes.

Asunto(s)

RESUMEN

An explanation is provided for the experimentally observed temperature dependence of the solubility and the solubility minimum of non-polar gases in water. The influence of solute size and solute-water attractive interactions on the solubility minimum temperature is investigated. The transfer of a non-polar solute from the ideal gas into water is divided into two steps: formation of a cavity in water with the size and shape of the solute and insertion of the solute in this cavity which is equivalent to 'turning on' solute-water attractive interactions. This two step process divides the excess chemical potential of the non-polar solute in water into repulsive and attractive contributions, respectively. The reversible work for cavity formation is modeled using an information theory model of hydrophobic hydration. Attractive contributions are calculated by modeling the water structure in the vicinity of non-polar solutes. These models make a direct connection between microscopic quantities and macroscopic observables. Moreover, they provide an understanding of the peculiar temperature dependences of the hydration thermodynamics from properties of pure water; specifically, bulk water density and the water oxygen-oxygen radial distribution function.

RESUMEN

Proteins can be denatured by pressures of a few hundred MPa. This finding apparently contradicts the most widely used model of protein stability, where the formation of a hydrophobic core drives protein folding. The pressure denaturation puzzle is resolved by focusing on the pressure-dependent transfer of water into the protein interior, in contrast to the transfer of nonpolar residues into water, the approach commonly taken in models of protein unfolding. Pressure denaturation of proteins can then be explained by the pressure destabilization of hydrophobic aggregates by using an information theory model of hydrophobic interactions. Pressure-denatured proteins, unlike heat-denatured proteins, retain a compact structure with water molecules penetrating their core. Activation volumes for hydrophobic contributions to protein folding and unfolding kinetics are positive. Clathrate hydrates are predicted to form by virtually the same mechanism that drives pressure denaturation of proteins.

Asunto(s)

RESUMEN

We address the molecular mechanism by which the haem-copper oxidases translocate protons. Reduction of O2 to water takes place at a haem iron-copper (CuB) centre, and protons enter from one side of the membrane through a 'channel' structure in the enzyme. Statistical-mechanical calculations predict bound water molecules within this channel, and mutagenesis experiments show that breaking this water structure impedes proton translocation. Hydrogen-bonded water molecules connect the channel further via a conserved glutamic acid residue to a histidine ligand of CuB. The glutamic acid side chain may have to move during proton transfer because proton translocation is abolished if it is forced to interact with a nearby lysine or arginine. Perturbing the CuB ligand structure shifts an infrared mode that may be ascribed to the O-H stretch of bound water. This is sensitive to mutations of the glutamic acid, supporting its connectivity to the histidine. These results suggest key roles of bound water, the glutamic acid and the histidine copper ligand in the mechanism of proton translocation.

Asunto(s)

RESUMEN

We present a statistical mechanical description of biomolecular hydration that accurately describes the hydrophobic and hydrophilic hydration of a model alpha-helical peptide. The local density of water molecules around a biomolecule is obtained by means of a potential-of-mean-force (PMF) expansion in terms of pair- and triplet-correlation functions of bulk water and dilute solutions of nonpolar atoms. The accuracy of the method is verified by comparing PMF results with the local density and site-site correlation functions obtained by molecular dynamics simulations of a model alpha-helix in solution. The PMF approach quantitatively reproduces all features of the peptide hydration determined from the molecular dynamics simulation. Regions of hydrophobic hydration near the C alpha and C beta atoms along the helix are well reproduced. The hydration of exposed polar groups at the N- and C-termini of the helix are also well described by the theory. A detailed comparison of the local hydration by means of site-site radial distribution functions evaluated with the PMF theory shows agreement with the molecular dynamics simulations. The formulation of this theory is general and can be applied to any biomolecular system. The accuracy, speed of computation, and local character of this theory make it especially suitable for studying large biomolecular systems.

Asunto(s)

RESUMEN

Cytochrome P450 enzymes are monooxygenases that contain a functional heme b group linked to a conserved cysteine with a thiolate bond. In the native state, the central iron atom is hexacoordinated with a covalently bound water molecule. The exclusion of solvent molecules from the active site is essential for efficient enzymatic function. Upon substrate binding, water has to be displaced from the active site to prevent electron uncoupling that results in hydrogen peroxide or water. In contrast to typical hemoproteins, the protein surface is not directly accessible from the heme of cytochromes P450. We postulate a two-state model in which a conserved arginine, stabilizing the heme propionate in all known cytochrome P450 crystal structures, changes from the initial, stable side-chain conformation to another rotamer (metastable). In this new state, a functional water channel (aqueduct) is formed from the active site to a water cluster located on the thiolate side of the heme, close to the protein surface. This water cluster communicates with the surface in the closed state and is partly replaced by the flipping arginine side chain in the open state, allowing water molecules to exit to the surface or to reaccess the active site. This two-state model suggests the presence of an exit pathway for water between the active site and the protein surface.

Asunto(s)

RESUMEN

A molecular model of poorly understood hydrophobic effects is heuristically developed using the methods of information theory. Because primitive hydrophobic effects can be tied to the probability of observing a molecular-sized cavity in the solvent, the probability distribution of the number of solvent centers in a cavity volume is modeled on the basis of the two moments available from the density and radial distribution of oxygen atoms in liquid water. The modeled distribution then yields the probability that no solvent centers are found in the cavity volume. This model is shown to account quantitatively for the central hydrophobic phenomena of cavity formation and association of inert gas solutes. The connection of information theory to statistical thermodynamics provides a basis for clarification of hydrophobic effects. The simplicity and flexibility of the approach suggest that it should permit applications to conformational equilibria of nonpolar solutes and hydrophobic residues in biopolymers.

RESUMEN

A three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine is proposed. This RNA, which was found using SELEX (Jenison et al., 1994), is 10,000 times more specific for theophylline (Kn = 320 nM) than for caffeine (KD = 3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a comparative molecular field analysis model (R2 = 0.93 for three components, with cross-validated R2 of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. On the basis of a model for the secondary structure (Jenison et al., 1994), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity are discussed.

Asunto(s)

RESUMEN

The local density of water molecules around a biomolecule is constructed from calculated two- and three-points correlation functions of polar solvents in water using a Potential-of-Mean-Force (PMF) expansion. As a simple approximation, the hydration of all polar (including charged) groups in a biomolecule is represented by the hydration of water oxygen in bulk water, and the effect of non-polar groups on hydration are neglected, except for excluded volume effects. Pair and triplet correlation functions are calculated by molecular dynamics simulations. We present calculations of the structural hydration for ideal A-DNA molecules with sequences [d(CG)5]2 and [d(C5G5)]2. We find that this method can accurately reproduce the hydration patterns of A-DNA observed in neutron diffraction experiments on oriented DNA fibers (P. Langan et al. J. Biomol. Struct. Dyn., 10, 489 (1992)).

Asunto(s)