Lorna Dougan

Single-Molecule Force Spectroscopy Measurements of Bond Elongation during a Bimolecular Reaction

It is experimentally challenging to directly obtain structural information of the transition state (TS), the high-energy bottleneck en route from reactants to products, for solution-phase reactions. Here, we use single-molecule experiments as well as high-level quantum chemical calculations to probe the TS of disulfide bond reduction, a bimolecular nucleophilic substitution (S N2) reaction. We use an atomic force microscope in force-clamp mode to apply mechanical forces to a protein disulfide bond and obtain force-dependent rate constants of the disulfide bond reduction initiated by a variety of nucleophiles. We measure distances to the TS or bond elongation (Delta x), along a 1-D reaction coordinate imposed by mechanical force, of 0.31 +/- 0.05 and 0.44 +/- 0.03 A for thiol-initiated and phosphine-initiated disulfide bond reductions, respectively. These results are in agreement with quantum chemical calculations, which show that the disulfide bond at the TS is longer in phosphine-initiated reduction than in thiol-initiated reduction. We also investigate the effect of solvent environment on the TS geometry by incorporating glycerol into the aqueous solution. In this case, the Delta x value for the phosphine-initiated reduction is decreased to 0.28 +/- 0.04 A whereas it remains unchanged for thiol-initiated reduction, providing a direct test of theoretical calculations of the role of solvent molecules in the reduction TS of an S N2 reaction. These results demonstrate that single-molecule force spectroscopy represents a novel experimental tool to study mechanochemistry and directly probe the sub-ångström changes in TS structure of solution-phase reactions. Furthermore, this single-molecule method opens new doors to gain molecular level understanding of chemical reactivity when combined with quantum chemical calculations.

Single homopolypeptide chains collapse into mechanically rigid conformations

Huntingtons disease is linked to the insertion of glutamine (Q) in the protein huntingtin, resulting in polyglutamine (polyQ) expansions that self-associate to form aggregates. While polyQ aggregation has been the subject of intense study, a correspondingly thorough understanding of individual polyQ chains is lacking. Here we demonstrate a single molecule force-clamp technique that directly probes the mechanical properties of single polyQ chains. We have made polyQ constructs of varying lengths that span the length range of normal and diseased polyQ expansions. Each polyQ construct is flanked by the I27 titin module, providing a clear mechanical fingerprint of the molecule being pulled. Remarkably, under the application of force, no extension is observed for any of the polyQ constructs. This is in direct contrast with the random coil protein PEVK of titin, which readily extends under force. Our measurements suggest that polyQ chains form mechanically stable collapsed structures. We test this hypothesis by disrupting polyQ chains with insertions of proline residues and find that their mechanical extensibility is sensitive to the position of the proline interruption. These experiments demonstrate that polyQ chains collapse to form a heterogeneous ensemble of conformations that are mechanically resilient. We further use a heat-annealing molecular dynamics protocol to extensively search the conformation space and find that polyQ can exist in highly mechanically stable compact globular conformations. The mechanical rigidity of these collapsed structures may exceed the functional ability of eukaryotic proteasomes, resulting in the accumulation of undigested polyQ sequences in vivo.

The structure of glycerol in the liquid state: a neutron diffraction study

Neutron diffraction coupled with hydrogen/deuterium isotopic substitution has been used to investigate the structure of the pure cryoprotectant glycerol in the liquid state at 298 K and 1 atm. The neutron diffraction data were used to constrain a 3 dimensional computational model that is experimentally relevant using the empirical potential structure refinement (EPSR) technique. These simulations lead to a model structure of the glycerol molecule that is consistent with the experimental data. Interestingly, from interrogation of this structure, it is found that the number of hydrogen bonds per molecule is larger than had previously been suggested. Furthermore, converse to previous work, no evidence for intra-molecular hydrogen bonds is found. These results highlight the importance and relevance of using experimental data to inform computational modelling of even simple liquid systems.

Molecular insight into the hydrogen bonding and micro-segregation of a cryoprotectant molecule.

Glycerol-water liquid mixtures are intriguing hydrogen-bonded systems and essential in many fields of chemistry, ranging from basic molecular research to widespread use in industrial and biomedical applications as cryoprotective solutions. Despite much research on these mixtures, the details of their microscopic structure are still not understood. One common notion is that glycerol acts to diminish the hydrogen bonding ability of water, a recurring hypothesis that remains untested by direct experimental approaches. The present work characterizes the structure of glycerol-water mixtures, across the concentration range, using a combination of neutron diffraction experiments and computational modeling. Contrary to previous expectations, we show that the hydrogen bonding ability of water is not diminished in the presence of glycerol. We show that glycerol-water hydrogen bonds effectively take the place of water-water hydrogen bonds, allowing water to maintain its full hydrogen bonding capacity regardless of the quantity of glycerol in the environment. We provide a quantitative measurement of all hydrogen bonding in the system and reveal a concentration range where a microsegregated, bipercolating liquid mixture exists in coexistence with a considerable interface region. This work highlights the role of hydrogen bonding connectivity rather than water structuring/destructuring effects in these important cryoprotective systems.

Preference for isolated water molecules in a concentrated glycerol-water mixture.

Neutron diffraction coupled with hydrogen/deuterium isotopic substitution has been used to investigate the structure of a concentrated glycerol water (4:1 mole fraction) solution. The neutron diffraction data were used to constrain a three-dimensional computational model that is experimentally relevant using the empirical potential structure refinement technique. From interrogation of this model, we find that glycerol-glycerol hydrogen bonding is largely unperturbed by the presence of water in the solution. We find that glycerol-water hydrogen bonding is prevalent, suggesting that water molecules effectively take the place of glycerol molecules in this concentrated solution. In contrast, we find that water-water hydrogen bonding is significantly perturbed. While the first coordination shell of water in the concentrated solution remains similar to that of pure water, water-water hydrogen bonding is greatly diminished beyond the first neighbor distance. Interestingly, the majority of water molecules exist as single monomers in the concentrated glycerol solution. The preference of isolated water molecules results in a solution that is well mixed with optimal glycerol-water hydrogen bonding. These results highlight the importance of preferential hydrogen bonding in aqueous solutions and suggest a mechanism for cryoprotection by which glycerol effectively hydrogen bonds with water, resulting in a disrupted hydrogen-bonded water network.

The hydrogen-bonding ability of the amino acid glutamine revealed by neutron diffraction experiments.

Hydrogen bonding between glutamine residues has been identified as playing an important role in the intermolecular association and aggregation of proteins. To establish the molecular mechanisms of glutamine interactions, neutron diffraction coupled with hydrogen/deuterium isotopic substitution in combination with computational modeling has been used to investigate the structure and hydration of glutamine in aqueous solution. The final structures obtained are consistent with the experimental data and provide insight into the hydrogen-bonding ability of glutamine. We find that the backbone of glutamine is able to coordinate more water molecules than the side chain, suggesting that charged groups on the glutamine molecule are more successful in attracting water than the dipole in the side chain. In both the backbone and the side chain, we find that the carbonyl groups interact more readily with water molecules than the amine groups. We find that glutamine-glutamine interactions are present, despite their low concentration in this dilute solution. This is evidenced through the occurrence of dimers of glutamine molecules in the solution, demonstrating the effective propensity of this molecule to associate through backbone-backbone, backbone-side chain, and side chain-side chain hydrogen bond interactions. The formation of dimers of glutamine molecules in such a dilute solution (30 mg/mL glutamine) may have implications in the aggregation of glutamine-rich proteins in neurological diseases where aggregation is prevalent.

Single-Molecule Force Spectroscopy Identifies a Small Cold Shock Protein as Being Mechanically Robust

Single-molecule force spectroscopy has emerged as a powerful approach to examine the stability and dynamics of single proteins. We have completed force extension experiments on the small cold shock protein B from Thermotoga maritima, using a specially constructed chimeric polyprotein. The proteins simple topology, which is distinct from the mechanically well-characterized β-grasp and immunoglobulin (Ig)-like folds, in addition to the wide range of structural homologues resulting from its ancient origin, provides an attractive model protein for single-molecule force spectroscopy studies. We have determined that the protein has mechanical stability, unfolding at greater than 70 pN at a pulling velocity of 100 nm s(-1). We reveal features of the unfolding energy landscape by measuring the dependence of the mechanical stability on pulling velocity, in combination with Monte Carlo simulations. We show that the cold shock protein has mechanically robust, yet malleable, features that may be important in providing the protein with stability and flexibility to function over a range of environmental conditions. These results provide insights into the relationship between the secondary structure and topology of a protein and its mechanical strength. This lays the foundation for the investigation of the effects of changes in environmental conditions on the mechanical and dynamic properties of cold shock proteins.

Stable Single α-Helices Are Constant Force Springs in Proteins

Background: Single α-helix (SAH) domains bridge two functional domains in proteins. Their force response is poorly understood. Results: Modeling and experiments show that SAH domains unfold non-cooperatively at low forces and maintain an approximately constant force as they unfold. Conclusion: SAH domains act as constant force springs. Significance: SAH domains are important mechanical elements in proteins. Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell.

What happens to the structure of water in cryoprotectant solutions

Cryoprotectant molecules are widely utilised in basic molecular research through to industrial and biomedical applications. The molecular mechanisms by which cryoprotectants stabilise and protect molecules and cells, along with suppressing the formation of ice, are incompletely understood. To gain greater insight into these mechanisms, we have completed an experimental determination of the structure of aqueous glycerol. Our investigation combines neutron diffraction experiments with isotopic substitution and computational modelling to determine the atomistic level structure of the glycerol-water mixtures, across the complete concentration range at room temperature. We examine the local structure of the system focusing on water structure. By comparing our data with that from other studies of cryoprotectant solutions, we attempt to find general rules for the action of cryoprotectants on water structure. We also discuss how these molecular scale interactions may be related to the macroscopic properties of the system.

Molecular self-assembly in a model amphiphile system

The physical origin of the large and negative excess entropy of mixing of alcohols and water remains controversial. In contrast to standard explanations that evoke concepts of water structuring, recent work has shown that, at ambient conditions, it can be quantitatively explained in terms of molecular scale partial demixing of the two components. Here, we estimate the negative excess entropy (DeltaS(E)) of aqueous methanol at low temperature and high pressure using experimentally-derived structural data and a recently introduced cluster model. On cooling to 190 K the cluster sizes increase, but the change in DeltaS(E), which according to this method of calculation depends on the surface area to volume ratio of the clusters, is not significant, suggesting that the topology of the clusters must change with decreased temperature. On compression the cluster sizes also increase, and DeltaS(E) is now positive, suggesting an even more pronounced change in cluster topology with increased pressure. This work suggests that it is the amphiphilic nature of a molecule that determines aggregation and self-assembly processes in aqueous solution. The results therefore give useful insight into the processes of cold and pressure denaturation of proteins.