Mapping Binding Epitopes and Allosteric Effects in Staph Enterotoxins

Abstract

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. In this work, we report an ancestral sequence reconstruction exercise targeting ancient nodes during the evolution of family-1 glycosidases and present the exceptional properties of a putative resurrected ancestor of bacterial and eukaryotic enzymes. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the $5500 reported crystallographic structures for $1400 modern glycosidases show a porphyrin ring in the enzyme. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein resurrection to reveal valuable but unusual protein features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation

“…An approach developed to overcome this limitation leverages solution-phase labeling of protein residues to encode the solvent accessibility and higher order structure of the protein into its mass. − MS is then utilized to assign the location of the modification. Due to their low sample requirements, rapid implementation, and applicability to proteins in membranes, disordered states, and complexes, these chemical labeling techniques have found a niche alongside the modern high-resolution structural techniques and have been widely utilized to address a diverse set of protein systems. − …”

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

“…Analyses of protein three-dimensional structure with mass spectrometry (MS) have traditionally been achieved by encoding the conformation of a protein into its mass through solution-phase chemical reactions. − The location and extent of labeling is dependent on reactant concentration, reactivity, and the solvent accessibility or protection factor of the participating chemical group. While these methodologies are frequently utilized, − the labeling reaction, digestion, and bioinformatic analyses required for these workflows set an upper limit on their throughput.…”

The path forward for protein footprinting, covalent labeling, and mass spectrometry‐based protein conformational analyses

“…The low sample requirements, rapid implementation, and broad applicability to proteins in membranes, disordered states, and complexes have led these chemical labeling-based MS techniques to be applied to a diverse set of protein systems. [13][14][15][16][17][18][19][20][21][22][23][24][25][26] The strengths of covalent labeling and the multiplexed analysis provided by MS make these techniques particularly well-equipped to elucidate changes in protein conformation of complex mixtures of proteins. This has led to their application directly to cells and living organisms.…”

“…The stability of these chemical modifications enables facile localization of labels to the peptide level and even to specific residues when bottom‐up proteomics workflows are employed. The low sample requirements, rapid implementation, and broad applicability to proteins in membranes, disordered states, and complexes have led these chemical labeling‐based MS techniques to be applied to a diverse set of protein systems 13–26 …”