Hydrophobic and ionic-interactions in bulk and confined water with implications for collapse and folding of proteins

Water and water-mediated interactions determine thermodynamic and kinetics of protein folding, protein aggregation and self-assembly in confined spaces. To obtain insights into the role of water in the context of folding problems, we describe computer simulations of a few related model systems. The dynamics of collapse of eicosane shows that upon expulsion of water the linear hydrocarbon chain adopts an ordered helical hairpin structure with 1.5 turns. The structure of dimer of eicosane molecules has two well ordered helical hairpins that are stacked perpendicular to each other. As a prelude to studying folding in confined spaces we used simulations to understand changes in hydrophobic and ionic interactions in nano droplets. Solvation of hydrophobic and charged species change drastically in nano water droplets. Hydrophobic species are localized at the boundary. The tendency of ions to be at the boundary where water density is low increases as the charge density decreases. Interaction between hydrophobic, polar, and charged residue are also profoundly altered in confined spaces. Using the results of computer simulations and accounting for loss of chain entropy upon confinement we argue and then demonstrate, using simulations in explicit water, that ordered states of generic amphiphilic peptide sequences should be stabilized in cylindrical nanopores

Properties of the free energy barriers for folding of the [alpha]-amylase inhibitor tendamistat

The goal of this work was to improve our understanding of the properties of the free energy barriers in protein folding. We used the small all-[beta]-sheet protein tendamistat as a model protein. Tendamistat contains two disulfide bridges and folds and unfolds in apparent two-state reactions. However, previous studies on disulfide variants demonstrated that tendamistat folds in at least two sequential steps with a high-energy intermediate. Elucidation of the properties of the free energy barriers by studying different tendamistat variants and various fragments provide a good insight into the underlying complexity of apparent two state folders. To obtain this information several approaches have been used in this work: we studied the combined influence of denaturant, temperature, structural variation and sodium sulfate on folding and stability; furthermore, we analysed the properties and stability of different fragments of tendamistat. Multiple perturbation analysis was used to gain information on the shape of the free energy barriers in tendamistat folding. Analysis of denaturant and temperature as perturbations revealed transition state movement according to the Hammond postulate. Hammond behaviour is more pronounced in the early transition state compared to the late transition state where only small transition state movement was observed. The results suggest that the early transition state is rather broad compared to the late transition state. These results emphasized the importance of multiple perturbation analysis to test the shape of the free energy barriers in protein folding. Determination of the activation parameters revealed less difference between both transition states. However, the denaturant dependence of the activation parameters of the transition states differs significantly. The results confirm our previous suggestion that the early transition state is broad and structurally less well defined, whereas the late transition state shows a rather narrow and structurally well-defined maximum. We further studied the effect of denaturant and structural variation on folding and stability. The results confirmed Hammond behaviour of the early transition state. To know more about structural properties of the transition states we determine

Design, Synthesis, And Conformational Studies Of Peptides Containing α,α-Disubstituted amino acids

Cα,α-disubstituted amino acids (ααAAs) are widely utilized to conformationally constrain peptides. Several pentapeptides containing dipropylglycine (Dpg) at alternating positions and their α-amino acid counterpart L-norvaline (Nva) analogues were synthesized to fully investigate the impact of Dpg on peptide backbone structure in aqueous solution. CD, VCD and NMR spectral analysis suggest that Dpg containing peptides adopt more ordered structures relative to their Nva containing analogues. The central residues (Ala, Thr, Tyr, Val) and the charged side-chains of Glu and Lys play important roles in the degree of peptide folding. Hydrophobic and branched residues (Val, Tyr) at the central position of the peptide produce greater folding as judged by CD and NMR. Temperature-dependent NMR analysis (Δδ/ ΔΤ ΝΗ) of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH2 suggests a series of i→i+3 hydrogen bonds between the N-terminal acetyl carbonyl and the Tyr3NH, and the Glu1 carbonyl and the Dpg4NH. The solution conformation of Ac-Glu-Dpg-Tyr-Dpg-Lys-NH2 calculated from NMR-derived constraints shows a 310-helical structure (two repetitive type-III β-turns) at residues 1-4, which is supported by 2D NMR, CD and VCD spectra. Analysis of NMR-derived models of these peptides suggest that there is a strong hydrophobic interaction of the pro-S propyl side chain of Dpg2 and the Tyr3 side-chain that may be a strong stabilizing force of the peptide folding in water. Cα,α-diisobutylglycine(Dibg) was synthesized via palladium catalyzed allylation reaction with an excellent overall yield and incorporated into various positions of a model β-hairpin peptide GHP in order to determine the effectiveness of the ααAAs as design elements in both the strand and turn portions of β-hairpins. CD and NMR data of Dibg containing peptides showed Dibg residue can contribute to the stability of the strand portion of a β-hairpin peptide and destabilize the β-turn in the GHP. The sheet stabilizing effect of Dibg may be due to the strong propensity of Dibg to have a fully extended conformation(φ/ψ = 180°). Several chiral amino esters were prepared with high enantioselectivity by alkylation of the corresponding Schiff bases under chiral phase-transfer condition. The enantiomeric excess of these chiral amino esters was efficiently determined by 19F-NMR analysis of the corresponding diastereomeric Mosher amides

Development & Application of Constant pH Molecular Dynamics (CPHMD ) for Investigating pH-mediated Transient Conformational States and Their Effects on Nucleic Acid & Protein Activity.

pH is a ubiquitous regulator of biological activity, with widespread impact ranging from its role in catalysis to carcinogenesis. Traditionally, a combination of biophysical and computational methods are used to measure pH-dependent activity profiles, and protonation equilibria (i.e. pKa values) of specific residues, and these data are used in conjunction with structural data to provide mechanistic understanding of pH-mediated biological function. More recent developments have also demonstrated the role of transient conformational states in a wide range of biological activities, which naturally leads to the question of how pH affects such transient states, and in turn, their resulting functional activity. In the study of biomolecular transient states, the detection limit is the key limitation of most experimental techniques. To bridge the gap in detection limit, we have developed an appropriate molecular dynamics based computational method, where protonation states are dynamically adjusted as a function of an external pH bath and the local environment surrounding the titrating site. Also known as the explicit solvent constant pH molecular dynamics (CPHMD^MSλD) framework, we use CPHMD^MSλD simulations and enhanced sampling methods to demonstrate the role of pH-regulated transient states in both nucleic acid and protein activity. First, we demonstrate the utility of CPHMD^MSλD simulations in conjunction with NMR experiments to characterize transiently populated Hoogsteen GC+ base pairs in DNA duplexes. The role of pH-dependent transient states is then generalized to RNA activity, including that of the catalytic mechanism of the hairpin ribozyme, where the existence of pH-dependent transient states can be used to reconcile a collection of seemingly inconsistent experimental observations in the literature. In addition, our CPHMD^MSλD simulations of proteins have elucidated the role of pH-dependent transient states in residues that are buried or occluded from solvent, including that of the pH-dependent optical properties of a cyan fluorescent protein mutant, where the existence of pH-dependent transient states can be used to explain its non-monotonic spectroscopic behavior.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113562/1/gbgoh_1.pd

Molecular Mechanism of Early Amyloid Self-Assembly Revealed by Computational Modeling

Protein misfolding followed by the formation of aggregates, is an early step in the cascade of conformational changes in a protein that underlie the development of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. Here, we demonstrate that the hairpin fold, a structure found in the early folding intermediates of amyloid b, induces morphological and stability changes in the aggregates of Aβ(14-23) peptide. We structurally characterized the interactions of monomer and hairpin using extended molecular dynamics (MD) simulations, which revealed a novel intercalated type complex. These finding suggest that folding patterns of amyloid proteins define the aggregation pathway. Computational analysis was then used to characterize the dimerization of full-length Aβ peptide and reveal their dynamic properties. Aβ dimers did not show β-sheet structures, as one may expect based on the known structures of Aβ fibrils, rather dimers are stabilized by hydrophobic interactions in the central hydrophobic regions. Comparison between Aβ40 and Aβ42 showed that overall, the dimers of both alloforms exhibit similar interaction strengths. However, the interaction patterns are significantly different. A novel aggregation pathway, able to describe aggregation at physiologically relevant concentrations, was elucidated when aggregation of amyloid proteins was performed in presence of surfaces. Computational analysis revealed that interaction of a monomer with the surface is accompanied by the structural transition of the monomer; which can then facilitate binding of another monomer and form a dimer. Compared to our previous data we observed an almost five-fold faster dimer formation. Further investigation of the surface-mediated aggregation revealed that lipid membranes promote aggregation of a-syn protein. MD simulations demonstrate that a-syn monomers change conformation upon interaction with the bilayers. On POPS, a-syn monomer protrudes from the surface. This conformation on POPS dramatically facilitates assembly of a dimer that remains stable over the entire simulation period. These findings are in line with experimental data. Overall, the studies described in this thesis provide the structural basis for the early stages of the misfolding and aggregation process of amyloid proteins

Macromolecular Interactions in West Nile Virus RNA-TIAR Protein Complexes and of Membrane Associated Kv Channel Peptides

Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replicatio