Pure Protein (United States)

Abstract Inverse protein folding methods have become central to the computational design of de novo proteins, but existing models struggle when tasked with generating high-affinity peptide binders. By combining peptide-specific finetuning with a novel decoding order strategy, we enhance pocket conditioning and enable more accurate sequence design for peptide-binding interfaces. Our approach delivers gains in computational metrics, increasing sequence recovery and improving in silico binder design success rate by 16% ™ 30%. In vitro validation finds that our method greatly improves the success rate of designing novel peptide agonists of the OPRM1 receptor, generating at least twice as many top-ranking agonists as the prevailing standard method ProteinMPNN.

In response to microbial infection, the nonclassical Ag-presenting molecule MHC class I-related protein 1 (MR1) presents secondary microbial metabolites to mucosal-associated invariant T (MAIT) cells. In this study, we further characterize the repertoire of ligands captured by MR1 produced in Hi5 (Trichoplusia ni) cells from Mycobacterium smegmatis via mass spectrometry. We describe the (to our knowledge) novel MR1 ligand photolumazine (PL)V, a hydroxyindolyl-ribityllumazine with four isomers differing in the positioning of a hydroxyl group. We show that all four isomers are produced by M. smegmatis in culture and that at least three can induce MR1 surface translocation. Furthermore, human MAIT cell clones expressing distinct TCR β-chains differentially responded to the PLV isomers, demonstrating that the subtle positioning of a single hydroxyl group modulates TCR recognition. This study emphasizes structural microheterogeneity within the MR1 Ag repertoire and the remarkable selectivity of MAIT cell TCRs.

Identification of many lichens require determination of their chemical constituents. Thin-layer chromatography is a chemical method for separating out chemicals by their solubility in organic solvents. We present here a tested, simplified protocol for performing thin-layer chromatography using locally available materials and reagents, based on solvent system C of standard lichen chromatographic protocols. Adapting Solvent System C, commercial-grade reagents were substituted and the protocol modified to be performed outside of a laboratory without the need of a special license for purchasing laboratory-grade chemicals and without special handling of hazardous chemicals and waste. Commercial-grade materials and chemicals were exchanged step-wise throughout the process. Trials completed using commercial-grade materials and chemicals were successfully compared to using labgrade materials and chemicals.

Peptide antigen presentation by major histocompatibility complex (MHC) class I proteins initiates CD8+ T cell-mediated immunity against pathogens and cancers. MHC I molecules typically bind peptides with 9 amino acids in length with both ends tucked inside the major A and F binding pockets. It has been known for a while that longer peptides can also bind by either bulging out of the groove in the middle of the peptide or by binding in a zigzag fashion inside the groove. In a recent study, we identified an alternative binding conformation of naturally occurring peptides from Toxoplasma gondii bound by HLA-A*02:01. These peptides were extended at the C terminus (PΩ) and contained charged amino acids not more than 3 residues after the anchor amino acid at PΩ, which enabled them to open the F pocket and expose their C-terminal extension into the solvent. Here, we show that the mechanism of F pocket opening is dictated by the charge of the first charged amino acid found within the extension. Although positively charged amino acids result in the Tyr-84 swing, amino acids that are negatively charged induce a not previously described Lys-146 lift. Furthermore, we demonstrate that the peptides with alternative binding modes have properties that fit very poorly to the conventional MHC class I pathway and suggest they are presented via alternative means, potentially including cross-presentation via the MHC class II pathway.

The U21 gene product from human herpesvirus 7 binds to and redirects class I major histocompatibility complex (MHC) molecules to a lysosomal compartment. The molecular mechanism by which U21 reroutes class I MHC molecules to lysosomes is not known. Here, we have reconstituted the interaction between purified soluble U21 and class I MHC molecules, suggesting that U21 does not require additional cellular proteins to interact with class I MHC molecules. Our results demonstrate that U21, itself predicted to contain an MHC class I-like protein fold, interacts tightly with class I MHC molecules as a tetramer, in a 4:2 stoichiometry. These observations have helped to elucidate a refined model describing the mechanism by which U21 escorts class I MHC molecules to the lysosomal compartment. In this report, we show that the human herpesvirus 7 (HHV-7) immunoevasin U21, itself a class I MHC-like protein, binds with high affinity to class I MHC molecules as a tetramer and escorts them to lysosomes, where they are degraded. While many class I MHC-like molecules have been described in detail, this unusual viral class I-like protein functions as a tetramer, associating with class I MHC molecules in a 4:2 ratio, illuminating a functional significance of homooligomerization of a class I MHC-like protein.