Biology subjects

Kopicki, J.-D.

Publications and source records attributed to Kopicki, J.-D..

3 recordsLinked to original sources

bioRxiv2025

X-ray Spectroscopy Meets Native Mass Spectrometry: Probing Gas-phase Protein Complexes

Gas-phase activation and dissociation studies of biomolecules, proteins and their non-covalent complexes using X-rays hold great promise for revealing new insights into the structure and function of biological samples. This is due to the unique properties of X-ray molecular interactions, such as site-specific and rapid ionization. In this perspective, we report and discuss the promise of first proof-of-principle studies of X-ray-induced dissociation of native biological samples ranging from small 17 kDa monomeric proteins up to large 808 kDa non-covalent protein assemblies conducted at a synchrotron (PETRA III) and a free-electron laser (FLASH2). A commercially available quadrupole time-of-flight mass spectrometer (Q-ToF2, Micromass/Waters), modified for high-mass analysis by MS Vision, was further adapted for integration with the open ports at the corresponding beamlines. The protein complexes were transferred natively into the gas phase via nano-electrospray ionization and subsequently probed by extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their folded state or following collision-induced activation in the gas phase. Depending on the size of the biomolecule and the activation method, protein fragmentation, dissociation, or enhanced ionization were observed. Additionally, an extension of the setup by ion mobility is described, which can serve as a powerful tool for structural separation of biomolecules prior to X-ray probing. The first experimental results are discussed in the broader context of current and upcoming X-ray sources, highlighting their potential for advancing structural biology in the future.

biochemistry↗

bioRxiv2021

Mapping the peptide binding groove of MHC class I

An essential element of adaptive immunity is the selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes on the cell surface. Using native mass spectrometry, we here analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule. This novel approach allows us to examine the binding properties of diverse peptides. The unique stability of our MHC class I even enables us to determine the binding affinity of complexes, which are suboptimally loaded with truncated or charge-reduced peptides. Notably, a unique erucamide adduct decouples affinity analysis from peptide identity alleviating issues usually attributed to clustering during electrospray ionization. We discovered that two anchor positions at the binding surface between MHC and peptide can be stabilized independently and further identify the contribution of other peptidic amino acids on the binding. We propose this as an alternative, likely universally applicable method to artificial prediction tools to estimate the binding strength of peptides to MHC class I complexes quickly and efficiently. This newly described MHC class I-peptide binding affinity quantitation represents a much needed orthogonal, independent approach to existing computational affinity predictions and has the potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy.

biophysics↗

bioRxiv2021

Dissociation of β2m from MHC Class I Triggers Formation of Noncovalent, Transient Heavy Chain Dimers

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin ({beta}2m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the 3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule co-localization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the 3 domain of one FHC binds to another FHC in a manner similar to the {beta}2m light chain. Significance StatementMHC class I molecules are cell surface transmembrane proteins with key functions in adaptive immunity against viral infections. The spatiotemporal organization of fully assembled MHC I at the cell surface and its function with respect to trans-interactions with T and NK cells has been studied in detail. By contrast, the consequences of peptide and {beta}2m dissociation yielding to formation of free heavy chains (FHC) have remained unclear. We have discovered that class I free heavy chains form distinct non-covalent dimers at the cell surface rather than non-specific clustering, and we have identified a dimerization interface mediated by the 3 domain. We propose that these non-covalent dimers are the basis of distinct signaling and endocytic sorting of MHC I FHC. This is to be explored in further work.

cell biology↗