Computational Graphics Software for Interactive Docking and Visualization of Ligand-Protein Complementarity.

The Dockeye software is designed to complement automated docking protocols by allowing the user's chemical know-how and experience of what makes for good protein-ligand binding, knowledge that is not easily encoded into automated algorithms, to guide the docking. It allows the interactive manipulation of the ligand placement against a protein target. Real-time intuitively comprehensible feedback about the location, spatial density, and the extent of both favorable and unfavorable atomic interactions between ligand and protein is provided through a carefully designed graphical object. It is also a tool for the graphical analysis of the interactions of known protein-ligand complexes. Comparative docking of 58 protein-ligand complexes with Dockeye and Autodock Vina shows how this software can be used synergistically with automated docking programs to significantly improve the task of discovery of ligand placement.

Entropy in molecular recognition by proteins.

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

DNA Conformational Changes Play a Force-Generating Role during Bacteriophage Genome Packaging.

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens as well as dsDNA bacteriophages-viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is instead an active component of the machinery. We carried out computational studies on dsDNA in the channels of viral portal proteins, and they reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer ("stretched") in regions of high negative electrostatic potential and shorter ("scrunched") in regions of high positive potential. These results suggest a mechanism that electrostatically couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis, and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA as the phosphate groups respond to the protein's electrostatic potential. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal, and dsDNA work synergistically to promote genome packaging.

Analysis of the size dependence of macromolecular crowding shows that smaller is better.

The aqueous milieu inside cells contains as much as 30-40% dissolved protein and RNA by volume. This large concentration of macromolecules is expected to cause significant deviations from solution ideality. In vivo biochemical reaction rates and equilibria might differ significantly from those measured in the majority of in vitro experiments that are performed at much lower macromolecule concentrations. Consequently crowding, a nonspecific phenomenon believed to arise from the large excluded volume of these macromolecules, has been studied extensively by experimental and theoretical methods. However, the relevant theory has not been applied consistently. When the steric effects of macromolecular crowders and small molecules like water and ions are treated on an equal footing, the effect of the macromolecules is opposite to that commonly believed. Large molecules are less effective at crowding than water and ions. There is also a surprisingly weak dependence on crowder size. Molecules of medium size, ∼5 Šradius, have the same effect as much larger macromolecules like proteins and RNA. These results require a reassessment of observed high-concentration effects and of strategies to mimic in vivo conditions with in vitro experiments.

Characterization of Cetyltrimethylammonium Bromide/Hexanol Reverse Micelles by Experimentally Benchmarked Molecular Dynamics Simulations.

Encapsulation of small molecules, proteins, and other macromolecules within the protective water core of reverse micelles is emerging as a powerful strategy for a variety of applications. The cationic surfactant cetyltrimethylammonium bromide (CTAB) in combination with hexanol as a cosurfactant is particularly useful in the context of solution NMR spectroscopy of encapsulated proteins. Small-angle X-ray and neutron scattering is employed to investigate the internal structure of the CTAB/hexanol reverse micelle particle under conditions appropriate for high-resolution NMR spectroscopy. The scattering profiles are used to benchmark extensive molecular dynamics simulations of this reverse micelle system and indicate that the parameters used in these simulations recapitulate experimental results. Scattering profiles and simulations indicate formation of homogeneous solutions of small approximately spherical reverse micelle particles at a water loading of 20 composed of ∼150 CTAB and 240 hexanol molecules. The 3000 waters comprising the reverse micelle core show a gradient of translational diffusion that reaches that of bulk water at the center. Rotational diffusion is slowed relative to bulk throughout the water core, with the greatest slowing near the CTAB headgroups. The 5 Šthick interfacial region of the micelle consists of overlapping layers of Br(-) enriched water, CTAB headgroups, and hexanol hydroxyl groups, containing about one-third of the total water. This study employs well-parametrized MD simulations, X-ray and neutron scattering, and electrostatic theory to illuminate fundamental properties of CTAB/hexanol reverse micelle size, shape, partitioning, and water behavior.

On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH < 0.8) shows that these report both on the local flexibility of the protein and on a different component of the conformational entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements.

A sharp thermal transition of fast aromatic-ring dynamics in ubiquitin.

Aromatic amino acid side chains have a rich role within proteins and are often central to their structure and function. Suitable isotopic-labelling strategies enable studies of sub-nanosecond aromatic-ring dynamics using solution NMR relaxation methods. Surprisingly, it was found that the three aromatic side chains in human ubiquitin show a sharp thermal dynamical transition at approximately 312 K. Hydrostatic pressure has little effect on the low-temperature behavior, but somewhat decreases the amplitude of motion in the high-temperature regime. Therefore, below the transition temperature, ring motion is largely librational. Above this temperature, a complete ring-rotation process that is fully consistent with a continuous diffusion not requiring the transient creation of a large activated free volume occurs. Molecular dynamics simulations qualitatively corroborate this view and reinforce the notion that the dynamical character of the protein interior has much more liquid-alkane-like properties than previously appreciated.

Banding of NMR-derived methyl order parameters: implications for protein dynamics.

Our understanding of protein folding, stability, and function has begun to more explicitly incorporate dynamical aspects. Nuclear magnetic resonance has emerged as a powerful experimental method for obtaining comprehensive site-resolved insight into protein motion. It has been observed that methyl-group motion tends to cluster into three "classes" when expressed in terms of the popular Lipari-Szabo model-free squared generalized order parameter. Here the origins of the three classes or bands in the distribution of order parameters are examined. As a first step, a Bayesian based approach, which makes no a priori assumption about the existence or number of bands, is developed to detect the banding of Oaxis2 values derived either from NMR experiments or molecular dynamics simulations. The analysis is applied to seven proteins with extensive molecular dynamics simulations of these proteins in explicit water to examine the relationship between O2 and fine details of the motion of methyl bearing side chains. All of the proteins studied display banding, with some subtle differences. We propose a very simple yet plausible physical mechanism for banding. Finally, our Bayesian method is used to analyze the measured distributions of methyl group motions in the catabolite activating protein and several of its mutants in various liganded states and discuss the functional implications of the observed banding to protein dynamics and function.

The remarkable hydration of the antifreeze protein Maxi: a computational study.

The long four-helix bundle antifreeze protein Maxi contains an unusual core for a globular protein. More than 400 ordered waters between the helices form a nano-pore of internal water about 150 Å long. Molecular dynamics simulations of hydrated Maxi were carried out using the CHARMM27 protein forcefield and the TIP3P water model. Solvation in the core and non-core first hydration shell was analyzed in terms of water-water H-bond distance-angle distributions. The core had an increased population of low-angle H-bonds between water pairs relative to bulk water. Enhancement of low angle H-bonds was particularly pronounced for water pairs at the interfaces between apolar and polar regions inside the protein core, characteristic of the anchored clathrate solvation structure seen previously in the ice-nuclei binding surfaces of type I, type III, and beta-helical antifreeze proteins. Anchored clathrate type solvation structure was not seen in the exterior solvation shell except around residues implicated in ice binding. Analysis of solvation dynamics using water residence times and diffusion constants showed that exterior solvation shell waters exchanged rapidly with bulk water, with no difference between ice-binding and non-binding residues. Core waters had about ten-fold slower diffusion than bulk water. Water residence times around core residues averaged about 8 ps, similar to those on the exterior surface, but they tended to exchange primarily with other core water, resulting in longer, >40 ps residence times within the core. Preferential exchange or diffusion of the water along the long axis of the water core of Maxi was not detected.

Microscopic insights into the NMR relaxation-based protein conformational entropy meter.

Conformational entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of conformational entropy are necessary for an understanding of its role but have been difficult to obtain. An empirical method that utilizes changes in conformational dynamics as a proxy for changes in conformational entropy has recently been introduced. Here we probe the microscopic origins of the link between conformational dynamics and conformational entropy using molecular dynamics simulations. Simulation of seven proteins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain conformational entropy. These results tend to validate the use of experimentally accessible measures of methyl motion--the NMR-derived generalized order parameters--as a proxy from which to derive changes in protein conformational entropy.

Intrinsic linear heterogeneity of amyloid ß protein fibrils revealed by higher resolution mass-per-length determinations.

Amyloid ß proteins spontaneously form fibrils in vitro that vary in their thermodynamic stability and in morphological characteristics such as length, width, shape, longitudinal twist, and the number of component filaments. It is vitally important to determine which variant best represents the type of fibril that accumulates in Alzheimer disease. In the present study, the nature of morphological variation was examined by dark-field and transmission electron microscopy in a preparation of seeded amyloid ß protein fibrils that formed at relatively low protein concentrations and exhibited remarkably high thermodynamic stability. The number of filaments comprising these fibrils changed frequently from two to six along their length, and these changes only became apparent when mass-per-length (MPL) determinations are made with sufficient resolution. The MPL results could be reproduced by a simple stochastic model with a single adjustable parameter. The presence of more than two primary filaments could not be discerned by transmission electron microscopy, and they had no apparent relationship to the longitudinal twist of the fibrils. However, the pitch of the twist was strongly affected by the pH of the negative stain. We conclude that highly stable amyloid fibrils may form in which a surprising amount of intrinsic linear heterogeneity may be obscured by MPL measurements of insufficient resolution, and by the negative stains used for imaging fibrils by electron microscopy.

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH.

The stability of bovine lactoperoxidase to denaturation by guanidinium-HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2 degrees C in the presence of 0.5, 1, and 2 M guanidinium-HCl, respectively. The extrapolated value for Tm in the absence of guanidinium-HCl is 73.7 degrees C, compared with 70.2 degrees C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium-HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 degrees C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164-1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.

Water in the half shell: structure of water, focusing on angular structure and solvation.

Water is a highly polar molecule, consisting of a very electronegative atom, oxygen, bonded to two weakly electropositive hydrogen atoms with two lone pairs of electrons. These features give water remarkable physical properties, some of which are anomalous, such as its lower density in the solid phase compared with the liquid phase. Its ability to serve as both a hydrogen bond donor and hydrogen bond acceptor governs its role as a solvent, a role that is of central interest for biological chemists. In this Account, we focus on water's properties as a solvent. Water dissolves a vast range of solutes with solubilities that range over 10 orders of magnitude. Differences in solubility define the fundamental dichotomy between polar, or hydrophilic, solutes and apolar, or hydrophobic, solutes. This important distinction plays a large part in the structure, stability, and function of biological macromolecules. The strength of hydrogen bonding depends on the H-O...O H-bond angle, and the angular distribution is bimodal. Changes in the width and frequency of infrared spectral lines and in the heat capacity of the solution provide a measure of the changes in the strength and distribution of angles of the hydrogen bonds. Polar solutes and inorganic ions increase the population of bent hydrogen bonds at the expense of the more linear population, while apolar solutes or groups have the opposite effect. We examine how protein denaturants might alter the solvation behavior of water. Urea has very little effect on water's hydrogen bond network, while guanidinium ions promote more linear hydrogen bonds. These results point to fundamental differences in the protein denaturation mechanisms of these molecules. We also suggest a mechanism of action for antifreeze (or thermal hysteresis) proteins: ordering of water around the surface of these proteins prior to freezing appears to interfere with ice formation.

Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity.

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4+ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.

Copy number evolution and its relationship with patient outcome-an analysis of 178 matched presentation-relapse tumor pairs from the Myeloma XI trial.

Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis-relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t(4;14) vs. stable pattern in t(11;14). CNA acquisition also differed between subtypes based on CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival (OS; hazard ratio (HR) 2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse (HR = 2.00; P = 0.021). There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.

Shape and evolution of thermostable protein structure.

Organisms evolved at high temperatures must maintain their proteins' structures in the face of increased thermal disorder. This challenge results in differences in residue utilization and overall structure. Focusing on thermostable/mesostable pairs of homologous structures, we have examined these differences using novel geometric measures: specifically burial depth (distance from the molecular surface to each atom) and travel depth (distance from the convex hull to the molecular surface that avoids the protein interior). These along with common metrics like packing and Wadell Sphericity are used to gain insight into the constraints experienced by thermophiles. Mean travel depth of hyperthermostable proteins is significantly less than that of their mesostable counterparts, indicating smaller, less numerous and less deep pockets. The mean burial depth of hyperthermostable proteins is significantly higher than that of mesostable proteins indicating that they bury more atoms further from the surface. The burial depth can also be tracked on the individual residue level, adding a finer level of detail to the standard exposed surface area analysis. Hyperthermostable proteins for the first time are shown to be more spherical than their mesostable homologues, regardless of when and how they adapted to extreme temperature. Additionally, residue specific burial depth examinations reveal that charged residues stay unburied, most other residues are slightly more buried and Alanine is more significantly buried in hyperthermostable proteins.

Protein pockets: inventory, shape, and comparison.

The shape of the protein surface dictates what interactions are possible with other macromolecules, but defining discrete pockets or possible interaction sites remains difficult. First, there is the problem of defining the extent of the pocket. Second, one has to characterize the shape of each pocket. Third, one needs to make quantitative comparisons between pockets on different proteins. An elegant solution to these problems is to sort all surface and solvent points by travel depth and then collect a hierarchical tree of pockets. The connectivity of the tree is determined via the deepest saddle points between each pair of neighboring pockets. The resulting pocket surfaces tessellate the entire protein surface, producing a complete inventory of pockets. This method of identifying pockets also allows one to easily compute important shape metrics, including the problematic pocket volume, surface area, and mouth size. Pockets are also annotated with their lining residue lists and polarity and with other residue-based properties. Using this tree and the various shape metrics pockets can be merged, grouped, or filtered for further analysis. Since this method includes the entire surface, it guarantees that any pocket of interest will be found among the output pockets, unlike all previous methods of pocket identification. The resulting hierarchy of pockets is easy to visualize and aids users in higher level analysis. Comparison of pockets is done by using the shape metrics, avoiding the complex shape alignment problem. Example applications show that the method facilitates pocket comparison along mutational or time-dependent series. Pockets from families of proteins can be examined using multiple pocket tree alignments to see how ligand binding sites or how other pockets have changed with evolution. Our method is called CLIPPERS for complete liberal inventory of protein pockets elucidating and reporting on shape.

Finding and characterizing tunnels in macromolecules with application to ion channels and pores.

We describe a new algorithm, CHUNNEL, to automatically find, characterize, and display tunnels or pores in proteins. The correctness and accuracy of the algorithm is verified on a constructed set of proteins and used to analyze large sets of real proteins. The verification set contains proteins with artificially created pores of known path and width profile. The previous benchmark algorithm, HOLE, is compared with the new algorithm. Results show that the major advantage of the new algorithm is that it can successfully find and characterize tunnels with no a priori guidance or clues about the location of the tunnel mouth, and it will successfully find multiple tunnels if present. CHUNNEL can also be used in conjunction with HOLE, with the former used toprime HOLE and the latter to track and characterize the pores. Analysis was conducted on families of membrane protein structures culled from the Protein Data Bank as well as on a set of transmembrane proteins with predicted membrane-aqueous phase interfaces, yielding the first completely automated examination of tunnels through membrane proteins, including tunnels that exit in the membrane bilayer.

Building alternate protein structures using the elastic network model.

We describe a method for efficiently generating ensembles of alternate, all-atom protein structures that (a) differ significantly from the starting structure, (b) have good stereochemistry (bonded geometry), and (c) have good steric properties (absence of atomic overlap). The method uses reconstruction from a series of backbone framework structures that are obtained from a modified elastic network model (ENM) by perturbation along low-frequency normal modes. To ensure good quality backbone frameworks, the single force parameter ENM is modified by introducing two more force parameters to characterize the interaction between the consecutive carbon alphas and those within the same secondary structure domain. The relative stiffness of the three parameters is parameterized to reproduce B-factors, while maintaining good bonded geometry. After parameterization, violations of experimental Calpha-Calpha distances and Calpha-Calpha-Calpha pseudo angles along the backbone are reduced to less than 1%. Simultaneously, the average B-factor correlation coefficient improves to R = 0.77. Two applications illustrate the potential of the approach. (1) 102,051 protein backbones spanning a conformational space of 15 A root mean square deviation were generated from 148 nonredundant proteins in the PDB database, and all-atom models with minimal bonded and nonbonded violations were produced from this ensemble of backbone structures using the SCWRL side chain building program. (2) Improved backbone templates for homology modeling. Fifteen query sequences were each modeled on two targets. For each of the 30 target frameworks, dozens of improved templates could be produced In all cases, improved full atom homology models resulted, of which 50% could be identified blind using the D-Fire statistical potential.