Gillian C. Lynch

Trimethylamine N-oxide influence on the backbone of proteins: An oligoglycine model

The study of organic osmolytes has been pivotal in demonstrating the role of solvent effects on the protein backbone in the folding process. Although a thermodynamic description of the interactions between the protein backbone and osmolyte has been well defined, the structural analysis of the effect of osmolyte on the protein backbone has been incomplete. Therefore, we have performed simulations of a peptide backbone model, glycine15, in protecting osmolyte trimethylamine N‐oxide (TMAO) solution, in order to determine the effect of the solution structure on the conformation of the peptide backbone. We show that the models chosen show that the ensemble of backbone structures shifts toward a more collapsed state in TMAO solution as compared with pure water solution. The collapse is consistent with preferential exclusion of the osmolyte caused by unfavorable interactions between osmolyte and peptide backbone. The exclusion is caused by strong triplet correlations of osmolyte, water, and peptide backbone. This provides a clear mechanism showing that even a modest concentration of TMAO forces the protein backbone to adopt a more collapsed structure in the absence of side chain effects. Proteins 2010.

Backbone additivity in the transfer model of protein solvation.

The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (ΔGtr) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N‐oxide (TMAO), were calculated from simulations of all atom models, and ΔGtr values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of −54 cal/mol/M compares quite favorably with −43 cal/mol/M determined experimentally.

Grand canonical ensemble molecular dynamics simulations: Reformulation of extended system dynamics approaches

The extended system Hamiltonian for carrying out grand canonical ensemble molecular dynamics simulations is reformulated. This new Hamiltonian includes a generalized treatment of the reference state partition function of the total chemical potential that reproduces the ideal gas behavior and various previous partitionings of ideal and excess terms. Initial calculations are performed on a system of Lennard–Jones particles near the triple point and on liquid water at room temperature.

Ion and solvent density distributions around canonical B-DNA from integral equations.

We calculate the water and ion spatial distributions around charged oligonucleotides using a renormalized three-dimensional reference interaction site theory coupled with the HNC closure. Our goal is to understand the balance between inter-DNA strand forces and solvation forces as a function of oligonucleotide length in the short strand limit. The DNA is considered in aqueous electrolyte solutions of 1 M KCl, 0.1 M KCl, or 0.1 M NaCl. The current theoretical results are compared to molecular dynamics (MD) simulations and experiments. It is found that the integral equation (IE) theory replicates the MD and the experimental results for the base-specific hydration patterns in both the major and the minor grooves. We are also able to discern characteristic structural pattern differences between Na(+) and K(+) ions. When compared to Poisson-Boltzmann methods, the IE theory, like simulation, predicts a richly structured ion environment, which is better described as multilayer rather than double layer.

Domain organization of membrane‐bound factor VIII

Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function.

Systematic Identification of Single Amino Acid Variants in Glioma Stem-Cell-Derived Chromosome 19 Proteins

Novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome. We have developed an approach, searching high quality shotgun proteomic data against an extended protein database, to identify expressed mutant proteoforms in glioma stem cell (GSC) lines. The systematic search of MS/MS spectra using PEAKS 7.0 as the search engine has recognized 17 chromosome 19 proteins in GSCs with altered amino acid sequences. The results were further verified by manual spectral examination, validating 19 proteoforms. One of the novel findings, a mutant form of branched-chain aminotransferase 2 (p.Thr186Arg), was verified at the transcript level and by targeted proteomics in several glioma stem cell lines. The structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma. Based on our initial findings, we believe that our approach presented could contribute to construct a more complete map of the human functional proteome.

Salt Effects on Surface-Tethered Peptides in Solution

The capability to manipulate proteins/peptide fragments at liquid-solid interfaces has led to tremendous applications in detectors and biotechnology. Therefore, understanding the detailed molecular behavior of proteins and peptides tethered on a hard material surface is an interesting and important topic. The inhomogeneity presented by surfaces as well as ions in the solution plays an important role in the thermodynamics and kinetics of the tethered proteins. In this study, we perform a series of molecular dynamics simulations of a pentapeptide RHSVV, a p53 epitope, tethered on a prepared microarray surface in various salt concentrations (0, 0.14, 0.5, and 1 M NaCl), as well as free in ionic solution (0, 0.5, and 1 M). The conformational space the tethered peptide visits largely overlaps with the free peptide in solution. However, surface tethering as well as the salt concentration changes both the thermodynamics and kinetics of the peptide. Frequent conformational changes are observed during the simulations and tend to be slowed down by both increasing the salt concentration and surface tethering. The local composition of ions at different salt concentrations is also compared between the tethered and free peptide.

Semi-grand canonical molecular dynamics simulation of bovine pancreatic trypsin inhibitor

Abstract In the quest to understand both the structural and thermodynamic facets of biomolecular–solvent systems semi-grand canonical ensemble molecular dynamics simulations of a protein in solution are performed. In these simulations only the water molecules in the system are allowed to fluctuate; the final number of water molecules is determined by the chemical potential. An unbiased sampling technique is used for the insertion/deletion procedure of the water molecules thereby providing a benchmark grand ensemble simulation of the hydration structure of proteins. Three different chemical potential simulations were carried out offering a direct route to thermodynamic information from a molecular dynamics simulation.

Norovirus escape from broadly neutralizing antibodies is limited to allostery-like mechanisms

The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development. ABSTRACT Ideal antiviral vaccines elicit antibodies (Abs) with broad strain recognition that bind to regions that are difficult to mutate for escape. Using 10 murine norovirus (MNV) strains and 5 human norovirus (HuNoV) virus-like particles (VLPs), we identified monoclonal antibody (MAb) 2D3, which broadly neutralized all MNV strains tested. Importantly, escape mutants corresponding to this antibody were very slow to develop and were distal to those raised against our previously studied antibody, A6.2. To understand the atomic details of 2D3 neutralization, we determined the cryo-electron microscopy (cryo-EM) structure of the 2D3/MNV1 complex. Interestingly, 2D3 binds to the top of the P domain, very close to where A6.2 binds, but the only escape mutations identified to date fall well outside the contact regions of both 2D3 and A6.2. To determine how mutations in distal residues could block antibody binding, we used molecular dynamics flexible fitting simulations of the atomic structures placed into the density map to examine the 2D3/MNV1 complex and these mutations. Our findings suggest that the escape mutant, V339I, may stabilize a salt bridge network at the P-domain dimer interface that, in an allostery-like manner, affects the conformational relaxation of the P domain and the efficiency of binding. They further highlight the unusual antigenic surface bound by MAb 2D3, one which elicits cross-reactive antibodies but which the virus is unable to alter to escape neutralization. These results may be leveraged to generate norovirus (NoV) vaccines containing broadly neutralizing antibodies. IMPORTANCE The simplest and most common way for viruses to escape antibody neutralization is by mutating residues that are essential for antibody binding. Escape mutations are strongly selected for by their effect on viral fitness, which is most often related to issues of protein folding, particle assembly, and capsid function. The studies presented here demonstrated that a broadly neutralizing antibody to mouse norovirus binds to an exposed surface but that the only escape mutants that arose were distal to the antibody binding surface. To understand this finding, we performed an in silico analysis that suggested that those escape mutations blocked antibody binding by affecting structural plasticity. This kind of antigenic region—one that gives rise to broadly neutralizing antibodies but that the virus finds difficult to escape from—is therefore ideal for vaccine development.

Solvation and Cavity Occupation in Biomolecules

BACKGROUNDnSolvation density locations are important for protein dynamics and structure. Knowledge of the preferred hydration sites at biomolecular interfaces and those in the interior of cavities can enhance understanding of structure and function. While advanced X-ray diffraction methods can provide accurate atomic structures for proteins, that technique is challenged when it comes to providing accurate hydration structures, especially for interfacial and cavity bound solvent molecules.nnnMETHODSnAdvances in integral equation theories which include more accurate methods for calculating the long-ranged Coulomb interaction contributions to the three-dimensional distribution functions make it possible to calculate angle dependent average solvent structure, accurately, around and inside irregular molecular conformations. The proximal radial distribution method provides another approximate method to determine average solvent structures for biomolecular systems based on a proximal or near neighbor solvent distribution that can be constructed from previously collected solvent distributions. These two approximate methods, along with all-atom molecular dynamics simulations are used to determine the solvent density inside the myoglobin heme cavity.nnnDISCUSSION AND RESULTSnMyoglobin is a good test system for these methods because the cavities are many and one is large, tens of Å(3), but is shown to have only four hydration sites. These sites are not near neighbors which implies that the large cavity must have more than one way in and out.nnnCONCLUSIONSnOur results show that main solvation sites are well reproduced by all three methods. The techniques also produce a clearly identifiable solvent pathway into the interior of the protein.nnnGENERAL SIGNIFICANCEnThe agreement between molecular dynamics and less computationally demanding approximate methods is encouraging. This article is part of a Special Issue entitled Recent developments of molecular dynamics.