Patricia Soto

Role of Water in Mediating the Assembly of Alzheimer Amyloid-β Aβ16–22 Protofilaments

The role of water in promoting the formation of protofilaments (the basic building blocks of amyloid fibrils) is investigated using fully atomic molecular dynamics simulations. Our model protofilament consists of two parallel beta-sheets of Alzheimer Amyloid-beta 16-22 peptides (Ac-K(16)-L(17)-V(18)-F(19)-F(20)-A(21)-E(22)-NH2). Each sheet presents a distinct hydrophobic and hydrophilic face and together self-assemble to a stable protofilament with a core consisting of purely hydrophobic residues (L(17), F(19), A(21)), with the two charged residues (K(16), E(22)) pointing to the solvent. Our simulations reveal a subtle interplay between a water mediated assembly and one driven by favorable energetic interactions between specific residues forming the interior of the protofilament. A dewetting transition, in which water expulsion precedes hydrophobic collapse, is observed for some, but not all molecular dynamics trajectories. In the trajectories in which no dewetting is observed, water expulsion and hydrophobic collapse occur simultaneously, with protofilament assembly driven by direct interactions between the hydrophobic side chains of the peptides (particularly between F-F residues). For those same trajectories, a small increase in the temperature of the simulation (on the order of 20 K) or a modest reduction in the peptide-water van der Waals attraction (on the order of 10%) is sufficient to induce a dewetting transition, suggesting that the existence of a dewetting transition in simulation might be sensitive to the details of the force field parametrization.

Oligomers of the Prion Protein Fragment 106-126 Are Likely Assembled from β-Hairpins in Solution, and Methionine Oxidation Inhibits Assembly without Altering the Peptide's Monomeric Conformation

A portion of the prion protein, PrP106-126, is highly conserved among various species and is thought to be one of the key domains involving amyloid formation of the protein. We used ion mobility spectrometry-mass spectrometry (IMS-MS) in conjunction with replica exchange molecular dynamics (REMD) to examine the monomeric and oligomeric structures of normal PrP106-126 and two nonaggregating forms of the peptide, an oxidized form in which both methionine residues are oxidized to methionine sulfoxide and a control peptide consisting of the same amino acids as PrP106-126 in a scrambled sequence. Our ion mobility and simulation data indicate the presence of a population of beta-hairpin monomers for the normal and oxidized peptides. This is supported by our CD data indicating that a monomer solution of the normal peptide contains approximately 46% beta-sheet and approximately 23% beta-turn content, in excellent agreement with our REMD simulations. Oligomerization was seen by IMS-MS for the normal peptide only, not the oxidized peptide or the control sequence. Both our IMS-MS and CD data suggest that this oligomerization results from the association of ordered beta-hairpin monomers rather than disordered monomers. Structural analysis shows that the normal and oxidized peptides have similar secondary and tertiary structural properties, suggesting that the inhibition of aggregation caused by methionine oxidation stems from mediating interpeptide interactions rather than by altering the peptides monomeric conformation. In contrast, an increase in alpha-helical and random coil structural components relative to the normal peptide might be responsible for the lack of observed aggregation of the control peptide.

Overhauser dynamic nuclear polarization and molecular dynamics simulations using pyrroline and piperidine ring nitroxide radicals.

The efficiency of Overhauser dynamic nuclear polarization (DNP) depends on the local dynamics modulating the dipolar coupling between the two interacting spins. By attaching nitroxide based spin labels to molecules and by measuring the (1)H DNP response of solvent water, information about the local hydration dynamics near the spin label can be obtained. However, there are two commonly used types of nitroxide ring structures; a pyrroline based and a piperidine based molecule. It is important to know when comparing different experiments, whether changes in DNP enhancements are due to changes in local hydration dynamics or because of the different spin label structures. In this study we investigate the key parameters affecting DNP signal enhancements for 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, a 5-membered ring nitroxide radical, and for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, a 6-membered ring nitroxide radical. Using X-Band DNP, field cycling relaxometry, and molecular dynamics simulations, we conclude that the key parameters affecting the DNP amplitude of the (1)H signal of water to be equal when using either nitroxide. Thus, experiments measuring hydration dynamics using either type of spin labels may be compared.

Aggregation of polyalanine in a hydrophobic environment

The dimerization of polyalanine peptides in a hydrophobic environment was explored using replica exchange molecular dynamics simulations. A nonpolar solvent (cyclohexane) was used to mimic, among other hydrophobic environments, the hydrophobic interior of a membrane in which the peptides are fully embedded. Our simulations reveal that while the polyalanine monomer preferentially adopts a beta-hairpin conformation, dimeric phases exist in an equilibrium between random coil, alpha-helical, beta-sheet, and beta-hairpin states. A thermodynamic characterization of the dimeric phases reveals that electric dipole-dipole interactions and optimal side-chain packing stabilize alpha-helical conformations, while hydrogen bond interactions favor beta-sheet conformations. Possible pathways leading to the formation of alpha-helical and beta-sheet dimers are discussed.

The effect of surface tethering on the folding of the src-SH3 protein domain

The effect of surface tethering on the folding mechanism of the src-SH3 protein domain was investigated using a coarse-grained Gō-type protein model. The protein was tethered at various locations along the protein chain and the thermodynamics and kinetics of folding were studied using replica exchange and constant temperature Langevin dynamics. Our simulations reveal that tethering in a structured part of the transition state can dramatically alter the folding mechanism, while tethering in an unstructured part leaves the folding mechanism unaltered as compared to bulk folding. Interestingly, there is only modest correlation between the tethering effect on the folding mechanism and its effect on thermodynamic stability and folding rates. We suggest locations on the protein at which tethering could be performed in single-molecule experiments so as to leave the folding mechanism unaltered from the bulk.

A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53–RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.