Jana Khandogin

Recent advances in implicit solvent-based methods for biomolecular simulations

Implicit solvent-based methods play an increasingly important role in molecular modeling of biomolecular structure and dynamics. Recent methodological developments have mainly focused on the extension of the generalized Born (GB) formalism for variable dielectric environments and accurate treatment of nonpolar solvation. Extensive efforts in parameterization of GB models and implicit solvent force fields have enabled ab initio simulation of protein folding to native or near-native structures. Another exciting area that has benefited from the advances in implicit solvent models is the development of constant pH molecular dynamics methods, which have recently been applied to the calculations of protein pK(a) values and the studies of pH-dependent peptide and protein folding.

Linking folding with aggregation in Alzheimer's beta-amyloid peptides.

Growing evidence suggests that the β-amyloid (Aβ) peptides of Alzheimers disease are generated in early endosomes and that small oligomers are the principal toxic species. We sought to understand whether and how the solution pH, which is more acidic in endosomes than the extracellular environment, affects the conformational processes of Aβ. Using constant pH molecular dynamics simulations of two model peptides, Aβ(1–28) and Aβ(10–42), we found that the folding landscape of Aβ is strongly modulated by pH and is most favorable for hydrophobically driven aggregation at pH 6. Thus, our theoretical findings substantiate the possibility that Aβ oligomers develop intracellularly before secretion into the extracellular milieu, where they may disrupt synaptic activity or act as seeds for plaque formation.

Exploring atomistic details of pH-dependent peptide folding

Modeling pH-coupled conformational dynamics allows one to probe many important pH-dependent biological processes, ranging from ATP synthesis, enzyme catalysis, and membrane fusion to protein folding/misfolding and amyloid formation. This work illustrates the strengths and capabilities of continuous constant pH molecular dynamics in exploring pH-dependent conformational transitions in proteins by revisiting an experimentally well studied model protein fragment, the C peptide from ribonuclease A. The simulation data reveal a bell-shaped pH profile for the total helix content, in agreement with experiment, and several pairs of electrostatic interactions that control the relative populations of unfolded and partially folded states of various helical lengths. The latter information greatly complements and extends that attainable by current experimental techniques. The present work paves the way for new and exciting applications, such as the study of pH-dependent molecular mechanism in the formation of amyloid comprising peptides from Alzheimers and Parkinsons diseases.

A Stable Hyponitrite-Bridged Iron Porphyrin Complex

The coupling of two nitric oxide (NO) molecules in heme active sites is an important contributor to the conversion of NO to nitrous oxide (N(2)O) by heme-containing enzymes. Several formulations for the presumed heme-Fe{N(2)O(2)}(n-) intermediates have been proposed previously, however, no crystal structures of heme-Fe{N(2)O(2)}(n-) systems have been reported to date. We report the first isolation and characterization of a stable bimetallic hyponitrite iron porphyrin, [(OEP)Fe](2)(mu-N(2)O(2)), prepared from the reaction of [(OEP)Fe](2)(mu-O) with hyponitrous acid. Density functional theoretical calculations were performed on the model compound [(porphine)Fe](2)(mu-N(2)O(2)) to characterize its electronic structure and properties.

Evidence for a catalytic dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae.

Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8295-8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS from Saccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in the S. cerevisiae HCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; a K act of 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A gives k cat, K AcCoA, and K alpha-KG values of 0.0031 s (-1), 13 muM, and 39 microM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained on V, with deuterium in the methyl of AcCoA. The pH dependence of k cat for E155A in the presence of formate gave a p K a of 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a p K a of about 6.7. The k cat for E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, giving k cat, K AcCoA, and K alpha-KG values of 0.039 s (-1), 33 microM, and 140 microM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 on V/ K AcCoA and V, respectively; the pH dependence of k cat was similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad.

Chapter 1 Molecular Simulations of pH-Mediated Biological Processes

Publisher Summary This chapter discusses the recent development of a pH-coupled molecular dynamics technique, called “continuous constant pH molecular dynamics” (CPHMD), and its applications to first principles pKa calculations and pH-coupled biological phenomena. Conformational dynamics and acid–base equilibria are microscopically coupled. However, traditional molecular dynamics simulations are performed with fixed protonation states. The development of CPHMD has improved the physical realism in molecular simulations. It has enabled, for the first time, quantitative pKa prediction for biological macromolecules on a first-principles level, thereby eliminating the need for ad hoc assignment of a protein dielectric constant and for a high-resolution structure as in the traditional Poisson–Boltzmann based approaches. Despite this success, the accuracy of the CPHMD method can be further improved through the continuing development of the underlying generalized Born (GB) implicit solvent model and its parameterization. The underestimation of pKa shifts for deeply buried residues may be reduced by using a molecular surface that accounts for the solvent excluded volume.

Modeling Protonation Equilibria In Biological Macromolecules

The stability and function of proteins are dependent on the charge states. For more than a decade, theoretical methods for the prediction of protonation equilibria in proteins have been based on a macroscopic description in which the dielectric response of protein to the fluctuating environment is modeled implicitly through an effective dielectric constant. Recently, constant pH molecular dynamics methods have been developed, which allow for an explicit coupling between the conformational dynamics and protonation equilibria in proteins. Of particular interest is the continuous constant pH method based on λ dynamics and GB implicit models. This method has enabled accurate and robust pK a predictions for proteins, and simulations of pH-coupled protein folding from first principles

Electrostatic Interaction In The Unfolded States Of Proteins

With recent recognition that the unfolded states of proteins play important and diverse roles in protein functions, some advances have been made in developing experimental techniques to help decipher residue-specific interactions. Here we present a molecular dynamics simulation based method that allows direct prediction of electrostatic interactions in the unfolded proteins under native conditions. The theoretical prediction is confirmed by measurements of pH-dependent folding free energies of a small model protein HP36.