Dataset Information

Hydrogen exchange soybean lipoxygenase

ABSTRACT: Defining specific pathways for efficient heat transfer from protein-solvent interfaces to their active sites represents one of the compelling and timely challenges in our quest for a physical description of the origins of enzyme catalysis. Enzymatic hydrogen tunneling reactions constitute excellent systems in which to validate experimental approaches to this important question, given the inherent temperature independence of quantum mechanical wave function overlap. Herein, we present the application of hydrogen deuterium exchange coupled to mass spectrometry toward the spatial resolution of protein motions that can be related to the chemical reaction within an enzyme active site. Employing the proton-coupled electron transfer reaction of soybean lipoxygenase as proof of principle, we first corroborate the impact of active site mutation on increased local flexibility and second, uncover a solvent-exposed loop, 15-34 Å from the reactive ferric center whose temperature-dependent motions are demonstrated to mirror the enthalpic barrier for catalytic C-H bond cleavage. A network that connects this surface loop to the active site is structurally identified and supported by changes in kinetic parameters that result from site-specific mutations.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Glycine Max

SUBMITTER: Adam Offenbacher

LAB HEAD: Judith P. Klinman

PROVIDER: PXD006234 | Pride | 2022-02-22

REPOSITORIES: Pride

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Publications

Hydrogen-Deuterium Exchange of Lipoxygenase Uncovers a Relationship between Distal, Solvent Exposed Protein Motions and the Thermal Activation Barrier for Catalytic Proton-Coupled Electron Tunneling.

Offenbacher Adam R AR Hu Shenshen S Poss Erin M EM Carr Cody A M CAM Scouras Alexander D AD Prigozhin Daniil M DM Iavarone Anthony T AT Palla Ali A Alber Tom T Fraser James S JS Klinman Judith P JP

ACS central science 20170609 6

Defining specific pathways for efficient heat transfer from protein-solvent interfaces to their active sites represents one of the compelling and timely challenges in our quest for a physical description of the origins of enzyme catalysis. Enzymatic hydrogen tunneling reactions constitute excellent systems in which to validate experimental approaches to this important question, given the inherent temperature independence of quantum mechanical wave function overlap. Herein, we present the applica ...[more]

PMID: 28691068

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Project description:Recent advances in software-driven glycopeptide identification in LC-MS/MS-based N-glycoproteomics have facilitated biochemical studies reporting thousands of intact N-glycopeptides, i.e. N-glycan-conjugated peptides, but the automated identification process remains to be scrutinized. Herein, we explore the efficiency of site-specific glycoprofiling using the PTM-centric search-engine Byonic relative to manual expert annotation. To allow an appropriately deep comparison, the study utilised typical glycoproteomics acquisition and data analysis strategies, but of a single glycoprotein, the uncharacterised N-glycosylated (Asn160, Asn268 and Asn302) human basigin. Detailed site-specific reference glycoprofiles of purified basigin were manually established using ion trap CID-MS/MS and high-resolution Q-Exactive Orbitrap HCD-MS/MS acquisition of tryptic N-glycopeptides and released N-glycans. The basigin N-glycosylation sites, which showed extensive micro- and macro-heterogeneity, were then glycoprofiled using Byonic with or without a background of complex peptides using Q-Exactive Orbitrap HCD-MS/MS data. The glycoprofiling efficiencies were assessed against the site-specific reference glycoprofiles and target and decoy proteome databases. The search criteria and confidence thresholds (Byonic scores) recommended by the vendor provided very high glycoprofiling accuracy and coverage (both >80%) and low peptide FDRs (<1%). The data complexity, search parameters including search space (proteome/glycome size), mass tolerance and peptide modifications, and confidence thresholds affected the glycoprofiling efficiency and analysis time. Automated identification of peptide modifications (methionine oxidation/carbamidomethylation) that coincide with monosaccharide mass differences (Fuc/Hex/HexNAc) and accurately distinguishing isobaric (Hex1NeuAc1-R/Fuc1NeuGc1-R) or near-isobaric (NeuAc1-R/Fuc2-R) monosaccharide sub-compositions remain challenging, arguing particular attention to such “difficult-to-identify” N-glycopeptides. The presented analysis provides valuable insights into automated glycopeptide identification; knowledge that facilitates further developments in FDR-based glycoproteomics.

Dataset Information

Hydrogen exchange soybean lipoxygenase

Publications

Hydrogen-Deuterium Exchange of Lipoxygenase Uncovers a Relationship between Distal, Solvent Exposed Protein Motions and the Thermal Activation Barrier for Catalytic Proton-Coupled Electron Tunneling.

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