Sheh-Yi Sheu

Energetics of hydrogen bonds in peptides

Hydrogen bonds and their relative strengths in proteins are of importance for understanding protein structure and protein motions. The correct strength of such hydrogen bonds is experimentally known to vary greatly from ≈5–6 kcal/mol for the isolated bond to ≈0.5–1.5 kcal/mol for proteins in solution. To estimate these bond strengths, here we suggest a direct novel kinetic procedure. This analyzes the timing of the trajectories of a properly averaged dynamic ensemble. Here we study the observed rupture of these hydrogen bonds in a molecular dynamics calculation as an alternative to using thermodynamics. This calculation is performed for the isolated system and contrasted with results for water. We find that the activation energy for the rupture of the hydrogen bond in a β-sheet under isolated conditions is 4.76 kcal/mol, and the activation energy is 1.58 kcal/mol for the same β-sheet in water. These results are in excellent agreement with observations and suggest that such a direct calculation can be useful for the prediction of hydrogen bond strengths in various environments of interest.

Tumor Cells Require Thymidylate Kinase to Prevent dUTP Incorporation during DNA Repair

The synthesis of dTDP is unique because there is a requirement for thymidylate kinase (TMPK). All other dNDPs including dUDP are directly produced by ribonucleotide reductase (RNR). We report the binding of TMPK and RNR at sites of DNA damage. In tumor cells, when TMPK function is blocked, dUTP is incorporated during DNA double-strand break (DSB) repair. Disrupting RNR recruitment to damage sites or reducing the expression of the R2 subunit of RNR prevents the impairment of DNA repair by TMPK intervention, indicating that RNR contributes to dUTP incorporation during DSB repair. We identified a cell-permeable nontoxic inhibitor of TMPK that sensitizes tumor cells to doxorubicin in vitro and in vivo, suggesting its potential as a therapeutic option.

Molecular dynamics of hydrogen bonds in protein-D2O: the solvent isotope effect.

We suggest that the H-bond in proteins not only mirrors the motion of hydrogen in its own atomistic setting but also finds its origin in the collective environment of the hydrogen bond in a global lattice of surrounding H2O molecules. This water lattice is being perturbed in its optimal entropic configuration by the motion of the H-bond. Furthermore, bonding interaction with the lattice drop the H-bond energy from some 5 kcal/mol for the pure protein in the absence of H2O, to some 1.6 kcal/mol in the presence of the H2O medium. This low value here is determined in a computer experiment involving MD calculations and is a value close to the generally accepted value for biological systems. In accordance with these computer experiments under ambient conditions, the H-bond energy is seriously depressed, hence confirming the subtle effect of the H2O medium directly interacting with the H-bond and permitting a strong fluxional behavior. Furthermore, water produces a very large change in the entropy of activation due to the hydrogen bond breakage, which affects the rate by as much as 2 orders of magnitude. We also observe that there is an entire ensemble of H-bond structures, rather than a single transition state, all of which contribute to this H-bond. Here the model is tested by changing to D2O as the surrounding medium resulting in a substantial solvent isotope effect. This demonstrates the important influence of the environment on the individual hydrogen bond.

Determination of protein surface hydration shell free energy of water motion: theoretical study and molecular dynamics simulation.

Water on protein surface plays a crucial role in the mechanistic aspects of biological processes; principally, this is characterized into two kinds of water molecules, biological water and bulk water. As compared to pure water, many aspects of the dynamics and structure of the surrounding water near the protein surface are much less understood. Evidence shows that those properties of the surrounding water induced by the presence of the biological systems differ from those of bulk water and that water has low mobility in the hydration shell. An intriguing question remains as to how to make a quantitative estimate of the hydration shell free energy when there is interaction between the protein and the hydration water. To explore this problem, we perform molecular dynamics simulation of the water motion in the hydration shell with respect to bulk water. A fractional Brownian motion theory combined with numerical simulation and a molecular dynamics simulation was developed. This theory was used to directly establish the connection between the dynamics of the protein surface water motion and the interaction between water and protein; this offers the possibility of determining the hydration shell free energy. In this study, we focused on water motion at the protein surface that is within a 4.4 Å layer, which is referred to as the hydration shell. We demonstrate that it actually follows a fractional Brownian motion. In this regime, a developed fractional Fokker-Planck equation, which is used to describe the dynamics of protein surface water motion, permits us to solve the mean first passage time of water molecules through the hydration shell. We then estimate the protein surface hydration shell free energy (HSFE), which depends on the barrier height of the hydration shell. For myoglobin, its HSFE is about 1.73 kcal/mol, and the accompanying activation entropy is 1.40R, where R is the gas constant. Corresponding reduced water mobility is observed for water surrounding myoglobin. In accord with the analysis of the radial distribution function, it is revealed that the effect of temperature on the HSFE is weak. The results show that the protein surface is wrapped by a shell of low mobility water motion and this hydration shell is dynamic rather than static.

Homology modeling and monoclonal antibody binding of the Der f 7 dust mite allergen

The group 7 allergens are important allergenic specificities for mite‐sensitive patients and may need to be incorporated into new diagnostic and therapeutic strategies. However, little is known about their biological and structural features. Position‐specific iterative BLAST showed that they had strong ancestral homology to two related families of lipid‐binding proteins, namely, the bactericidal permeability‐increasing (BPI) proteins and the odorant‐binding protein. A three‐dimensional model of Der f 7 made with the Phyre and SWISS‐MODEL homology‐modeling servers showed a close match with the human BPI coordinates used for its construction. The binding of the monoclonal antibody HD12 known to block IgE binding could be blocked by the linear sequence (46GILDF50) with a critical role for L48 or F50. These hydrophobic residues were located on a surface loop of the model. The properties of Der f 7 that can be deduced from the model provide avenues for further characterizing these allergens, their IgE binding structures and biological properties that can enhance allergenicity.

Gated escaping of ligand out of protein

We construct a new gating model and develop a new theory to study the escaping process of a ligand out of a spherical cavity with a puncture (or gate) on the surface. The gate undulation can be regulated by any time-dependent function and the motion of the ligand inside the spherical cavity is mapped into a two-dimensional entropy potential surface. Hence the driving force of our model is entropy only. For a static gate, the escaping process corresponds to climbing a two-dimensional entropy barrier. When the gate open angle is small, the escaping rate is proportional to the square of the opening angle. The prefactor of the escaping rate constant depends on the curvature of the entropy potential surface. For coherent gating, the survival time depends not only on the gate undulation frequency but also on how the initial state is defined. On the escaping from protein, our escaping rate shows it is qualitatively consistent with the experimental result of ligand recombination in myoglobin.

Kramers theory of chemical reactions in a slowly adjusting environment

When describing the reaction dynamics in a slowly relaxing environment, one has to include slow nonreactive modes of the environment in an explicit consideration along with the “chemical” mode intrinsically responsible for the chemical transformation. This is done within the framework of the Kramers approach to condensed phase chemical reaction dynamics. The problem is studied under the condition of high friction of the nonreactive mode (slow adjustment) while friction of the chemical mode covers the whole range from weak to high friction. It is found that the reaction dynamics and, hence, the kinetics depend strongly on the strength of the coupling of the reactive and the nonreactive modes. For strong mode coupling the rate constant monotonically decreases with the increase of the friction of the chemical mode. Such behavior is quite distinctive from one for fast adjustment of the environment when the rate constant demonstrates a turnover behavior. Turnover behavior takes place for moderate strength mode...

Hydrogen Bonds in Membrane Proteins

Hydrogen bonds are essential tie points inside protein structures. They undergo dynamic rupture and rebonding processes on the time scale of tens of picoseconds. Proteins can partially rearrange during such ruptures. In previous work, we performed molecular dynamics simulations of these fluctuating hydrogen bonds. This indicated long-range entropy and energy contributions extending far into the liquid environment. The results showed that the binding of a given hydrogen bond is much reduced as a result of these interactions in water, as is required for biological activity and in very good confirmation of known experimental results. The larger water environment directly interacts with the hydrogen bond essentially due to long-range molecular interactions. Such a substantial lowering of the energy of the hydrogen bond in water brings it into the range of activation by many biological processes ( Sheu et al. Chem. Phys. Lett. 2008 , 462 , 1 - 5 ). Thus, the water medium profoundly increases the rate. Furthermore, very large entropic changes are associated with the rupture of hydrogen bonds in water, whereas no such effects are seen for the isolated molecule. Interestingly, such an increase in rates in water is still accompanied by a large negative change in entropy in the extended solvent environment, and this reduces the rate by some 2 orders of magnitude. Recent molecular dynamics experiments in D(2)O substantiate this model and show a large solvent isotope effect. In this work, we used lipids as the environment for the hydrogen bond and discovered that the energy is also reduced from that found in the isolated molecule, but not as far as in water. On the other hand, we found that no entropy penalty exists for breaking the hydrogen bond in lipids, as seen for water. These two effects compensate, even though the energy is some 2 times larger. The entropic penalty is reduced such that the rate is higher than in water despite the higher energy. This is a significant result for understanding the reactivity and dynamics of proteins in lipids. It should be noted that these are very important solvent effects on entropies and free energies that are not usually reflected in statistical thermodynamic computations for reactants and products. The very long-range effect of the solvent makes substantial contributions to kinetic rate constants and is readily evaluated in this kinetic method. To ignore these long-range environmental effects on the entropy can lead to very spurious results when calculating rates of protein mobilities. Hence, the results not only agree very well with the known hydrogen-bond energies directly as a result of various environmental factors, but even correctly predict a phase transition in the lipid.

Stochastic gating influence on the kinetics of diffusion-limited reactions

We study how the kinetics of diffusion-influenced reactions is modified when the reactivity of species fluctuates in time (stochastically gated) with emphasis on the many-particle aspect of the problem. Because of the fact that the dynamics of ligand binding to proteins originally motivated the problem, it is considered in that context. Recently, Zhou and Szabo [J. Phys. Chem. 100, 2597 (1996)] have demonstrated many-particle effects in the problem and found that the kinetics of reaction between a gated protein with a large number of ligands significantly differs from that between a protein and gated ligands. With our approach, the difference between the kinetics of ligand-gated and protein-gated reactions appears formally the same as the difference between the target and trapping problems despite the origin of the corresponding effects and their manifestations are distinctly different. A simple approximate method to treat the many-particle effects is proposed. The theory is applied to a particular two-st...

Structural Insights into the Catalytic Active Site and Activity of Human Nit2/ω-Amidase KINETIC ASSAY AND MOLECULAR DYNAMICS SIMULATION

Background: Human Nit2/ω-amidase is a putative tumor suppressor. Results: Both the catalytic triad and loop 116–128 of hNit2 play an essential role in the enzyme-substrate binding and enzymatic activity. Conclusion: The results of MD simulations are consistent with the kinetic analysis obtained with substrates α-ketoglutaramate and succinamate. Significance: This work provides the basis for new areas of research into tumor glutamine metabolism and hyperammonemic diseases. Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/ω-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/ω-amidase was determined with α-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116–128 deletion to validate the role of key residues and the 116–128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/ω-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/ω-amidase. A catalytic triad and the loop residues 116–128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.