Submillisecond Atomistic Molecular Dynamics Simulations Reveal Hydrogen Bond-Driven Diffusion of a Guest Peptide in Protein-RNA Condensate.

Liquid-liquid phase separation mediated by proteins and/or nucleic acids is believed to underlie the formation of many distinct condensed phases, or membraneless organelles, within living cells. These condensates have been proposed to orchestrate a variety of important processes. Despite recent advances, the interactions that regulate the dynamics of molecules within a condensate remain poorly understood. We performed accumulated 564.7 µs all-atom molecular dynamics (MD) simulations (system size ∼200k atoms) of model condensates formed by a scaffold RNA oligomer and a scaffold peptide rich in arginine (Arg). These model condensates contained one of three possible guest peptides: the scaffold peptide itself or a variant in which six Arg residues were replaced by lysine (Lys) or asymmetric dimethyl arginine (ADMA). We found that the Arg-rich peptide can form the largest number of hydrogen bonds and bind the strongest to the scaffold RNA in the condensate, relative to the Lys- and ADMA-rich peptides. Our MD simulations also showed that the Arg-rich peptide diffused more slowly in the condensate relative to the other two guest peptides, which is consistent with a recent fluorescence microscopy study. There was no significant increase in the number of cation-π interactions between the Arg-rich peptide and the scaffold RNA compared to the Lys-rich and ADMA-rich peptides. Our results indicate that hydrogen bonds between the peptides and the RNA backbone, rather than cation-π interactions, play a major role in regulating peptide diffusion in the condensate.

Thioamide Analogues of MHC I Antigen Peptides.

Short, synthetic peptides that are displayed by major histocompatibility complex I (MHC I) can stimulate CD8 T cells in vivo to destroy virus-infected or cancer cells. The development of such peptides as vaccines that provide protective immunity, however, is limited by rapid proteolytic degradation. Introduction of unnatural amino acid residues can suppress MHC I antigen proteolysis, but the modified peptides typically display lower affinity for MHC I and/or diminished ability to activate CD8 T cells relative to native antigen. Here, we report a new strategy for modifying MHC I antigens to enhance resistance to proteolysis while preserving MHC I affinity and T cell activation properties. This approach, replacing backbone amide groups with thioamides, was evaluated in two well-characterized antigens presented by HLA-A2, a common human MHC I. For each antigen, singly modified thioamide analogues retained affinity for HLA-A2 and activated T cells specific for the native antigen, as measured via interferon-γ secretion. In each system, we identified a highly potent triply substituted thioamide antigen ("thio-antigen") that displayed substantial resistance to proteolytic cleavage. Collectively, our results suggest that thio-antigens may represent a general and readily accessible source of potent vaccine candidates that resist degradation.

Trimer-to-Monomer Disruption Mechanism for a Potent, Protease-Resistant Antagonist of Tumor Necrosis Factor-α Signaling.

Aberrant tumor necrosis factor-α (TNFα) signaling is associated with many inflammatory diseases. The homotrimeric quaternary structure of TNFα is essential for receptor recognition and signal transduction. Previously, we described an engineered α/ß-peptide inhibitor that potently suppresses TNFα activity and resists proteolysis. Here, we present structural evidence that both the α/ß-peptide inhibitor and an all-α analogue bind to a monomeric form of TNFα. Calorimetry data support a 1:1 inhibitor/TNFα stoichiometry in solution. In contrast, previous cocrystal structures involving peptide or small-molecule inhibitors have shown the antagonists engaging a TNFα dimer. The structural data reveal why our inhibitors favor monomeric TNFα. Previous efforts to block TNFα-induced cell death with peptide inhibitors revealed that surfactant additives to the assay conditions cause a more rapid manifestation of inhibitory activity than is observed in the absence of additives. We attributed this effect to a loose surfactant TNFα association that lowers the barrier to trimer dissociation. Here, we used the new structural data to design peptide inhibitors bearing a surfactant-inspired appendage intended to facilitate TNFα trimer dissociation. The appendage modified the time course of protection from cell death.

Formation of versus Recruitment to RNA-Rich Condensates: Controlling Effects Exerted by Peptide Side Chain Identity.

Liquid-liquid phase separation (LLPS), the spontaneous formation of contiguous liquid phases with distinct compositions, has been long known in chemical systems and more recently recognized as a ubiquitous feature of cell biology. We describe a system involving biologically relevant components, synthetic peptides, and total yeast RNA, that has enabled us to explore factors that underlie phase separation. Coulombic complementarity between a cationic peptide and anionic RNA is necessary but not sufficient for formation of a condensed phase in our system. In addition to a net positive charge, the peptide must present the proper type of cationic moiety. Guanidinium groups, as found in the Arg side chain, support phase separation, but ammonium groups, as found in the Lys side chain, or dimethylguanidinium groups, as found in post-translationally modified Arg side chains, do not support phase separation in our system. However, the cationic groups that do not support phase separation via interaction with RNA can nevertheless enable recruitment to a condensed phase, which reveals that the network of forces governing condensed phase formation can differ from the network of forces governing recruitment to such a phase. We introduce a new method for measuring the concentrations of components in condensed phases based on fluorine-containing additives and 19F NMR.

Wrinkling and folding of thin films by viscous stress.

We examine the buckling of a thin elastic film floating on a viscous liquid layer which is itself supported on a prestretched rubber sheet. Releasing the prestretch in the rubber induces a viscous stress in the liquid, which in turn induces a compressive stress in the elastic film, leading to buckling. Unlike many previous studies on wrinkling of floating films, the buckling process in the present study is dominated by viscous effects whereas gravitational effects are negligible. An approximate shear lag model predicts the evolution of the stress profile in the unbuckled film that depends on three parameters: the rate at which the prestretch is released, the thickness of the liquid layer, and the length of the elastic film. A linear perturbation analysis is developed to predict the wavelength of wrinkles. Numerical simulations are conducted to predict nonlinear evolution of the wrinkle wavelength and amplitude. Experiments using elastic polymer films and viscous polymer liquids show trends that are qualitatively consistent with the predictions although quantitatively, the experimentally-observed wrinkle wavelengths are longer than predicted. Although this article is focused only on small-strain wrinkling behavior, we show that application of large nominal strains (on the order of 100%) leads to sharply localized folds. Thus this approach may be useful for developing buckled features with high aspect ratio on surfaces.