Anthony Hazel

Thermodynamics of Deca-alanine Folding in Water

The determination of the folding dynamics of polypeptides and proteins is critical in characterizing their functions in biological systems. Numerous computational models and methods have been developed for studying structure formation at the atomic level. Due to its small size and simple structure, deca-alanine is used as a model system in molecular dynamics (MD) simulations. The free energy of unfolding in vacuum has been studied extensively using the end-to-end distance of the peptide as the reaction coordinate. However, few studies have been conducted in the presence of explicit solvent. Previous results show a significant decrease in the free energy of extended conformations in water, but the α-helical state is still notably favored over the extended state. Although sufficient in vacuum, we show that end-to-end distance is incapable of capturing the full complexity of deca-alanine folding in water. Using α-helical content as a second reaction coordinate, we deduce a more descriptive free-energy landscape, one which reveals a second energy minimum in the extended conformations that is of comparable free energy to the α-helical state. Equilibrium simulations demonstrate the relative stability of the extended and α-helical states in water as well as the transition between the two states. This work reveals both the necessity and challenge of determining a proper reaction coordinate to fully characterize a given process.

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin

Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.

Computed Free Energies of Peptide Insertion into Bilayers are Independent of Computational Method

We show that the free energy of inserting hydrophobic peptides into lipid bilayer membranes from surface-aligned to transmembrane inserted states can be reliably calculated using atomistic models. We use two entirely different computational methods: high temperature spontaneous peptide insertion calculations as well as umbrella sampling potential-of-mean-force (PMF) calculations, both yielding the same energetic profiles. The insertion free energies were calculated using two different protein and lipid force fields (OPLS protein/united-atom lipids and CHARMM36 protein/all-atom lipids) and found to be independent of the simulation parameters. In addition, the free energy of insertion is found to be independent of temperature for both force fields. However, we find major difference in the partitioning kinetics between OPLS and CHARMM36, likely due to the difference in roughness of the underlying free energy surfaces. Our results demonstrate not only a reliable method to calculate insertion free energies for peptides, but also represent a rare case where equilibrium simulations and PMF calculations can be directly compared.

Folding free energy landscapes of β-sheets with non-polarizable and polarizable CHARMM force fields

Molecular dynamics (MD) simulations of peptides and proteins offer atomic-level detail into many biological processes, although the degree of insight depends on the accuracy of the force fields used to represent them. Protein folding is a key example in which the accurate reproduction of folded-state conformations of proteins and kinetics of the folding processes in simulation is a longstanding goal. Although there have been a number of recent successes, challenges remain in capturing the full complexity of folding for even secondary-structure elements. In the present work, we have used all-atom MD simulations to study the folding properties of one such element, the C-terminal β-hairpin of the B1 domain of streptococcal protein G (GB1). Using replica-exchange umbrella sampling simulations, we examined the folding free energy of two fixed-charge CHARMM force fields, CHARMM36 and CHARMM22*, as well as a polarizable force field, the CHARMM Drude-2013 model, which has previously been shown to improve the folding properties of α-helical peptides. The CHARMM22* and Drude-2013 models are in rough agreement with experimental studies of GB1 folding, while CHARMM36 overstabilizes the β-hairpin. Additional free-energy calculations show that small adjustments to the atomic polarizabilities in the Drude-2013 model can improve both the backbone solubility and folding properties of GB1 without significantly affecting the models ability to properly fold α-helices. We also identify a non-native salt bridge in the β-turn region that overstabilizes the β-hairpin in the C36 model. Finally, we demonstrate that tryptophan fluorescence is insufficient for capturing the full β-hairpin folding pathway.

Correction to: Computed Free Energies of Peptide Insertion into Bilayers are Independent of Computational Method

The original version of the article unfortunately contained an error in NIH support grant number RO1-GM74639 in the Acknowledgements section. The correct grant number is RO1-GM74637. This has been corrected with this erratum.

Diverse Protein-Folding Pathways and Functions of β-Hairpins and β-Sheets

One of the most common fundamental secondary-structure elements of proteins is the β-sheet. Folding of β-sheets into various structures, e.g., β-hairpins, β-barrels, and amyloids, is believed to provide the energy required to drive various processes, including membrane-protein insertion into and translocation across the outer membrane of Gram-negative bacteria. The folding of the β-hairpin has also been proposed to function as a conformational switch in some systems. In this chapter, we review the contributions of molecular dynamics simulations to resolving the energetics of β-hairpin folding, the mechanics of β-barrel insertion into the membrane by the BAM complex, the dynamics of β-hairpin conformational switching, and the structures of disease-causing amyloids.

Thermodynamics of β-Structures from Molecular Dynamics Simulations

β-Sheets are some of the most common secondary structure motifs in proteins, and are important for mediating protein-protein interactions through their association. This association can also lead to the aggregation of misfolded proteins into β-pleated-sheets in neurodegenerative disorders like amyloidosis. The folding pathway from random coil to β-sheet usually involves two competing process: (1) the collapse of a hydrophobic core, and (2) the formation of intrapeptide hydrogen bonds. It has been proposed, and shown computationally, that the hydrophobic core collapse precedes hydrogen bond formation. In this study we examine the thermodynamics of β-hairpin formation for the GB1 domain of protein G with molecular dynamics simulations by calculating a two-dimensional free energy surface in both vacuum and explicit water using as our reaction coordinates (1) the radius of gyration of the hydrophobic core and (2) the number of native hydrogen bonds, corresponding to the two aforementioned folding processes, respectively. We also compare the results of different versions of the CHARMM force field, namely CHARMM22, CHARMM22/CMAP, CHARMM22∗ and CHARMM36. Finally, we show how these methods can be applied to other β-structures in vivo, namely β-helix structures in the outer membrane of Gram-negative bacteria.