Taking the Monte-Carlo gamble: How not to buckle under the pressure!

Consistent buckling distortions of a large membrane patch (200 × 200 Å) are observed during molecular dynamics (MD) simulations using the Monte-Carlo (MC) barostat in combination with a hard Lennard-Jones (LJ) cutoff. The buckling behavior is independent of both the simulation engine and the force field but requires the MC barostat-hard LJ cutoff combination. Similar simulations of a smaller patch (90 × 90 Å) do not show buckling, but do show a small, systematic reduction in the surface area accompanied by ~1 Å thickening suggestive of compression. We show that a mismatch in the way potentials and forces are handled in the dynamical equations versus the MC barostat results in a compressive load on the membrane. Moreover, a straightforward application of elasticity theory reveals that a minimal compression of the linear dimensions of the membrane, inversely proportional to the edge length, is required for buckling, explaining this differential behavior. We recommend always using LJ force or potential-switching when the MC barostat is employed to avoid undesirable membrane deformations.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Harmonizing Experimental Data with Modeling to Predict Membrane Protein Insertion in Yeast.

Membrane proteins must adopt their proper topologies within biological membranes, but achieving the correct topology is compromised by the presence of marginally hydrophobic transmembrane helices (TMHs). In this study, we report on a new model membrane protein in yeast that harbors two TMHs fused to an unstable nucleotide-binding domain. Because the second helix (TMH2) in this reporter has an unfavorable predicted free energy of insertion, we employed established methods to generate variants that alter TMH2 insertion free energy. We first found that altering TMH2 did not significantly affect the extent of protein degradation by the cellular quality control machinery. Next, we correlated predicted insertion free energies from a knowledge-based energy scale with the measured apparent free energies of TMH2 insertion. Although the predicted and apparent insertion energies showed a similar trend, the predicted free-energy changes spanned an unanticipated narrow range. By instead using a physics-based model, we obtained a broader range of free energies that agreed considerably better with the magnitude of the experimentally derived values. Nevertheless, some variants still inserted better in yeast than predicted from energy-based scales. Therefore, molecular dynamics simulations were performed and indicated that the corresponding mutations induced conformational changes within TMH2, which altered the number of stabilizing hydrogen bonds. Together, our results offer insight into the ability of the cellular quality control machinery to recognize conformationally distinct misfolded topomers, provide a model to assess TMH insertion in vivo, and indicate that TMH insertion energy scales may be limited depending on the specific protein and the mutation present.

Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot.

Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 µs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 µs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.