The occurrence of linear cholesterol-recognition motifs in alpha-helical transmembrane domains has long been debated. Here, we demonstrate the ability of a genetic algorithm guided by coarse-grained molecular dynamics simulations---a method coined evolutionary molecular dynamics (evo-MD)---to directly resolve the sequence which maximally attracts/sorts cholesterol within a single-pass alpha-helical transmembrane domain (TMDs). We illustrate that the evolutionary landscape of cholesterol attraction in membrane proteins is characterized by a sharp, well-defined global optimum. Surprisingly, this optimal solution features an unusual short hydrophobic block, consisting of typically only eight short chain hydrophobic amino acids, surrounded by three successive lysines. Owing to the membrane thickening effect of cholesterol, cholesterol-enriched ordered phases favor TMDs characterized by a long rather than a short hydrophobic length. However, this short hydrophobic pattern evidently offers a pronounced net advantage for the binding of free cholesterol in both coarse-grained and atomistic simulations. Attraction is mediated by the unique ability of cholesterol to snorkel within the hydrophobic core of the membrane and thereby shield deeply located lysines from the unfavorable hydrophobic surrounding. Since this mechanism of attraction is of a thermodynamic nature and is not based on molecular shape specificity, a large diversity of sub-optimal cholesterol attracting sequences can exist. The puzzling sequence variability of proposed linear cholesterol-recognition motifs is thus consistent with sub-optimal, unspecific binding of cholesterol. Importantly, since evo-MD uniquely enables the targeted design of recognition motifs for distinct fluid lipid membranes, we foresee wide applications for evo-MD in the biological and biomedical fields.

The Tat system translocates folded proteins across energy-transducing prokaryotic membranes. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was tolerated. Length-variation in TatB clearly destabilized TatBC-containing complexes, indicating that the 12-residues-length is crucial for Tat component interactions and translocon assembly. Metal tagging transmission electron microscopy revealed the dimensions of TatA assemblies, which prompted molecular dynamics simulations. These showed that interacting TMHs of larger TatA assemblies can thin the membrane together with laterally aligned tilted APHs that generate a deep V-shaped groove. The conserved hydrophobic mismatch may thus be important for membrane destabilization during Tat transport, and the exact length of 12 hydrophobic residues could be a compromise between functionality and proton leakage minimization.

The aggregation of peptides into amyloid fibrils is linked to ageing-related diseases, such as Alzheimer's disease and type 2 diabetes. Interfaces, particularly those with large nanostructured surface areas, can affect the kinetics of peptide aggregation, ranging from a complete inhibition to strong acceleration. While a number of physiochemical parameters determine interface effects, we here focus on the role of nanoparticle curvature for the aggregation of the amyloidogenic peptides Aβ40, NNFGAIL, GNNQQNY and VQIYVK. Nanoparticles (NPs) provided a surface for peptide monomers to adsorb, enabling the nucleation into oligomers and fibril formation. High surface curvature, however, destabilized prefibrillar structures, providing an explanation for inhibitory effects on fibril growth. Thioflavin T (ThT) fluorescence assays as well as dynamic light scattering (DLS), atomic force microscopy (AFM) and electron microscopy experiments revealed NP size-dependent effects on amyloid fibril formation, with differences between the peptides. While 5 nm gold NPs (AuNP-5) retarded or inhibited the aggregation of most peptides, larger 20 nm gold NPs (AuNP-20) tended to accelerate peptide aggregation. Molecular dynamics (MD) studies demonstrated that NPs' ability to catalyze or inhibit oligomer formation was influenced by the oligomer stability at curved interfaces which was lower at more highly curved surfaces. Differences in the NP effects for the peptides resulted from the peptide properties (size, aggregation propensity) and concomitant surface binding affinities. The results can be applied to the design of future nanostructured materials for defined applications.