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Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases. [ABSTRACT FROM AUTHOR]

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/μm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future. [ABSTRACT FROM AUTHOR]

Short peptides reflecting major histocompatibility complex (MHC) class I (MHC‐I) epitopes frequently lack sufficient immunogenicity to induce robust antigen (Ag)‐specific CD8+ T cell responses. In the current work, it is demonstrated that position‐scanning peptide libraries themselves can serve as improved immunogens, inducing Ag‐specific CD8+ T cells with greater frequency and function than the wild‐type epitope. The approach involves displaying the entire position‐scanning library onto immunogenic nanoliposomes. Each library contains the MHC‐I epitope with a single randomized position. When a recently identified MHC‐I epitope in the glycoprotein gp70 envelope protein of murine leukemia virus (MuLV) is assessed, only one of the eight positional libraries tested, randomized at amino acid position 5 (Pos5), shows enhanced induction of Ag‐specific CD8+ T cells. A second MHC‐I epitope from gp70 is assessed in the same manner and shows, in contrast, multiple positional libraries (Pos1, Pos3, Pos5, and Pos8) as well as the library mixture give rise to enhanced CD8+ T cell responses. The library mixture Pos1‐3‐5‐8 induces a more diverse epitope‐specific T‐cell repertoire with superior antitumor efficacy compared to an established single mutation mimotope (AH1‐A5). These data show that positional peptide libraries can serve as immunogens for improving CD8+ T‐cell responses against endogenously expressed MHC‐I epitopes. [ABSTRACT FROM AUTHOR]

The article presents a study that explores how lyophilized, thermostable Spike or receptor-binding domain immunogenic liposomes induce protective immunity against SARS-CoV-2 in mice. It mentions about the assessment of low-dose immunization with lyophilized nanoparticles decorated with recombinant SARS-CoV-2 antigens.