Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide-empty intermediate.

The cell biology and biochemistry of peptide exchange on major histocompatibility complex class I (MHC-I) proteins are of great interest in the study of immunodominance, which requires iterative optimization of peptide affinity, and cross-presentation of pathogen and tumor antigens, in which endogenous peptides are exchanged for exogenous ones. Even though several methods exist to catalyze peptide exchange on recombinant MHC-I proteins, the cellular conditions and mechanisms allowing for peptide exchange in vivo remain unclear. Here, we demonstrate that low pH, as present in endosomes, indeed triggers peptide exchange, and we dissect the individual steps of the exchange reaction.

Rapid peptide exchange on MHC class I by small molecules elucidates dynamics of bound peptide.

Complexes of peptides with recombinant major histocompatibility complex class I molecules (rpMHCs) are an important tool for T cell detection, isolation, and activation in cancer immunotherapy. The rapid preparation of rpMHCs is aided by peptide exchange, for which several technologies exist. Here, we show peptide exchange with small-molecule alcohols and demonstrate that they accelerate the dissociation of pre-bound peptides, creating a novel method for rapid production of rpMHCs and increasing the understanding of the conformational flexibility of the MHC-bound peptides.

Opening opportunities for K determination and screening of MHC peptide complexes.

An essential element of adaptive immunity is selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes. Using native mass spectrometry, we analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule and, due to its unique stability, we determine binding affinities of complexes loaded with truncated or charge-reduced peptides. We find that the two anchor positions can be stabilized independently, and we further analyze the contribution of additional amino acid positions to the binding strength.

Peptide-MHC I complex stability measured by nanoscale differential scanning fluorimetry reveals molecular mechanism of thermal denaturation.

Recombinant major histocompatibility complex class I molecules are used in diagnostic and therapeutic approaches in cancer immunotherapy, with many studies exploring their binding to antigenic peptides. Current techniques for kinetic peptide binding studies are hampered by high sample consumption, low throughput, interference with protein stability, and/or high background signal. Here, we validate nanoscale differential scanning fluorimetry (nanoDSF), a method using the tryptophan fluorescence of class I molecules, for class I/peptide binding, and we use it to determine the molecular mechanism of the thermal denaturation of HLA-A*02:01.

High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules.

Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8 T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive.

Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells.

The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the α and α helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01.

Production and Multiplication of Native Compost Fungal Activator by Using Different Substrates and Its Influence on Growth and Development of Capsicum chinensis Jacq. "Bhut Jolokia".

In vitro experiment was carried out to see the effect of saw dusts of Pinus kesiya, Shorea robusta, and Callicarpa arborea on Trichoderma harzianum, isolate TH-13 mass production, along with its biotization effect on Capsicum chinensis Jacq. "Bhut Jolokia." Early mycelium initiation (2 days) occurred in S.