21:T462,How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-β-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.22:T53f,Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of -1PRF is coordinated by a 'slippery' sequence in the transcript, an adjacent RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop reduce the efficiency of -1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of -1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

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