Roles of cosolvents on protein stability

The function of a protein is determined by its three-dimensional structure which emerges from the delicate balance of forces involving atoms of the protein and the solvent. This balance can be perturbed by changing temperature, pressure, pH and by adding organic molecules known as cosolvents to the solution. Despite the wide use of cosolvents to perturb protein structures in the lab and in living systems, their molecular mechanisms are still not well established. Understanding these mechanisms is a problem of substantial interest, with potential application to the design of new drugs to target proteins. In this dissertation, we probe the role of two major cosolvents, urea and trimethylamine N-oxide (TMAO) at atomic level. Urea is widely used as a denaturant in the lab to destabilize native protein conformations. However, the atomic mechanism of this molecule remains a question of debate. To unravel its molecular mechanism, explicit all-atom molecular dynamics simulations of unrestrained and extended poly-alanine and poly-leucine dimers are performed. Consistent with experimental results, we find that the large non-polar side chain of leucine is affected by urea whereas backbone atoms and alanine’s side chain are not. Urea is found to occupy positions between leucine’s side chains that are not accessible to water. This accounts for extra Lennard-Jones bonds between urea and side chains that favors the unfolded state. These bonds compete with urea-solvent interactions that favor the folded state. The sum of these two energetic terms provides the enthalpic driving force for unfolding. It is shown here that this enthalpy correlates with the potential of mean force of poly-leucine dimers. To provide insights into the stabilizing mechanisms TMAO on protein structures, microsecond all-atom molecular dynamics simulations of peptides and replica exchange molecular dynamics simulations (REMD) of the Trp-cage miniprotein are performed. Most previous studies have focused on the effect of this osmolyte on protein backbone. Our results are consistent with these studies as we show that TMAO induces the backbone to adopt compact conformations. However, it is shown that effects of TMAO on the backbone are not dominant. In particular, TMAO\u27s effect on the backbone is overcompensated by its destabilizing effect on the hydrophobic core: non-polar peptides and residues forming the hydrophobic core of the Trp-cage protein adopt more extended conformations in solutions containing TMAO. It is found that a main interaction that can stabilize folded proteins are charge-charge interactions. In light of these results, we propose that competing effects of TMAO on hydrophobic and charge-charge interactions account for its net stabilizing role on proteins

A Systematic Test of Receptor Binding Kinetics for Ligands in Tumor Necrosis Factor Superfamily by Computational Simulations

Ligands in the tumor necrosis factor (TNF) superfamily are one major class of cytokines that bind to their corresponding receptors in the tumor necrosis factor receptor (TNFR) superfamily and initiate multiple intracellular signaling pathways during inflammation, tissue homeostasis, and cell differentiation. Mutations in the genes that encode TNF ligands or TNFR receptors result in a large variety of diseases. The development of therapeutic treatment for these diseases can be greatly benefitted from the knowledge on binding properties of these ligand–receptor interactions. In order to complement the limitations in the current experimental methods that measure the binding constants of TNF/TNFR interactions, we developed a new simulation strategy to computationally estimate the association and dissociation between a ligand and its receptor. We systematically tested this strategy to a comprehensive dataset that contained structures of diverse complexes between TNF ligands and their corresponding receptors in the TNFR superfamily. We demonstrated that the binding stabilities inferred from our simulation results were compatible with existing experimental data. We further compared the binding kinetics of different TNF/TNFR systems, and explored their potential functional implication. We suggest that the transient binding between ligands and cell surface receptors leads into a dynamic nature of cross-membrane signal transduction, whereas the slow but strong binding of these ligands to the soluble decoy receptors is naturally designed to fulfill their functions as inhibitors of signal activation. Therefore, our computational approach serves as a useful addition to current experimental techniques for the quantitatively comparison of interactions across different members in the TNF and TNFR superfamily. It also provides a mechanistic understanding to the functions of TNF-associated cell signaling pathways

Pseudoinverse Determination of the Circulation in Prydz Bay and its Adjacent Open Ocean, Antarctica

An inverse model is used to infer the circulation in Prydz Bay and its adjacent open ocean using hydrographic data obtained by the cruise of the 7th Chinese National Antarctic Research Expedition (CHINARE-7), 1990/91. Barotropic components are found to be strong in the study area, esp. at the Antarctic Divergence, and from a whole view, the velocity is rather small. In the open ocean, the flow is quasizonal, but outside the bay it shows a tendency of pressing onto the shelf from surface to bottom, and a feature of intensification just east of Fram Bank. We suggest here be the most important place to detect the possibility of the Antarctic Bottom Water formation. The meridionial profiles of the distribution indicate a strong (relative to the ambient) core and a slope-trapped part into the bargain. In the southeastern part of the bay, there seems to exist a strong coastal current flowing westward. The computed upwelling centers are mainly situated in the west of the study region, as agrees quite well with the early hydrographic observations and the areas of high krill biomass

A condition for the formation of Antarctic Bottom Water in Prydz Bay, Antarctica

Through pseudoinverse inference of the circulation in Prydz Bay and its adjacent open ocean during January to March 1981, and comparing the results with that of 1991, we find that when the polar easterly hence the east wind drift is strong and extends its influence north of the slope, it is difficult for the Circumpolar Deep Water (CDW) to upwell onto the shelf, and consequently the Antarctic Bottom Water (AABW) cannot form in the bay by way of mixing scheme of Foster and Carmack (1976). However, when the East Wind Drift weakens the confines itself over the shelf, the westerly current will press on the slope and revolve anticyclonically so long as it is fairly strong. Such an anti-cyclonical pattern manifests itself mainly in the lower layer, and as a result, it will make the CDW upwell onto the shelf, providing an essential prerequisite for the formation of the AABW. We have analyzed this phenomenon from a dynamical view, and pointed out that the law of heat conduction accounts for its formation, in which the planetary and topographical beta effects play major roles

Using Coarse-Grained Simulations to Characterize the Mechanisms of Protein–Protein Association

The formation of functionally versatile protein complexes underlies almost every biological process. The estimation of how fast these complexes can be formed has broad implications for unravelling the mechanism of biomolecular recognition. This kinetic property is traditionally quantified by association rates, which can be measured through various experimental techniques. To complement these time-consuming and labor-intensive approaches, we developed a coarse-grained simulation approach to study the physical processes of protein–protein association. We systematically calibrated our simulation method against a large-scale benchmark set. By combining a physics-based force field with a statistically-derived potential in the simulation, we found that the association rates of more than 80% of protein complexes can be correctly predicted within one order of magnitude relative to their experimental measurements. We further showed that a mixture of force fields derived from complementary sources was able to describe the process of protein–protein association with mechanistic details. For instance, we show that association of a protein complex contains multiple steps in which proteins continuously search their local binding orientations and form non-native-like intermediates through repeated dissociation and re-association. Moreover, with an ensemble of loosely bound encounter complexes observed around their native conformation, we suggest that the transition states of protein–protein association could be highly diverse on the structural level. Our study also supports the idea in which the association of a protein complex is driven by a “funnel-like” energy landscape. In summary, these results shed light on our understanding of how protein–protein recognition is kinetically modulated, and our coarse-grained simulation approach can serve as a useful addition to the existing experimental approaches that measure protein–protein association rates

A computational study of co-inhibitory immune complex assembly at the interface between T cells and antigen presenting cells.

The activation and differentiation of T-cells are mainly directly by their co-regulatory receptors. T lymphocyte-associated protein-4 (CTLA-4) and programed cell death-1 (PD-1) are two of the most important co-regulatory receptors. Binding of PD-1 and CTLA-4 with their corresponding ligands programed cell death-ligand 1 (PD-L1) and B7 on the antigen presenting cells (APC) activates two central co-inhibitory signaling pathways to suppress T cell functions. Interestingly, recent experiments have identified a new cis-interaction between PD-L1 and B7, suggesting that a crosstalk exists between two co-inhibitory receptors and the two pairs of ligand-receptor complexes can undergo dynamic oligomerization. Inspired by these experimental evidences, we developed a coarse-grained model to characterize the assembling of an immune complex consisting of CLTA-4, B7, PD-L1 and PD-1. These four proteins and their interactions form a small network motif. The temporal dynamics and spatial pattern formation of this network was simulated by a diffusion-reaction algorithm. Our simulation method incorporates the membrane confinement of cell surface proteins and geometric arrangement of different binding interfaces between these proteins. A wide range of binding constants was tested for the interactions involved in the network. Interestingly, we show that the CTLA-4/B7 ligand-receptor complexes can first form linear oligomers, while these oligomers further align together into two-dimensional clusters. Similar phenomenon has also been observed in other systems of cell surface proteins. Our test results further indicate that both co-inhibitory signaling pathways activated by B7 and PD-L1 can be down-regulated by the new cis-interaction between these two ligands, consistent with previous experimental evidences. Finally, the simulations also suggest that the dynamic and the spatial properties of the immune complex assembly are highly determined by the energetics of molecular interactions in the network. Our study, therefore, brings new insights to the co-regulatory mechanisms of T cell activation

Driving β‑Strands into Fibrils

In this work we study contributions of mainchain and side chain atoms to fibrillization of polyalanine peptides using all-atom molecular dynamics simulations. We show that the total number of hydrogen bonds in the system does not change significantly during aggregation. This emerges from a compensatory mechanism where the formation of one interpeptide hydrogen bond requires rupture of two peptide–water bonds, leading to the formation of one extra water–water bond. Since hydrogen bonds are mostly electrostatic in nature, this mechanism implies that electrostatic energies related to these bonds are not minimized during fibril formation. Therefore, hydrogen bonds do not drive fibrillization in all-atom models. Nevertheless, they play an important role in this process since aggregation without the formation of interpeptide hydrogen bonds accounts for a prohibitively large electrostatic penalty (∼9.4 kJ/mol). Our work also highlights the importance of using accurate models to describe chemical bonds since Lennard-Jones and electrostatic contributions of different chemical groups of the protein and solvent are 1 order of magnitude larger than the overall enthalpy of the system. Thus, small errors in modeling these interactions can produce large errors in the total enthalpy of the system