A novel opportunities for the structural characterization of the biomolecular assemblies

Introduction

Covalent labeling and chemical cross-linking in combination with mass spectrometry is a powerful approach for structural biologists to approach proteins" structure, interaction and dynamic. Recently, the toolbox of covalent labeling techniques has been expanded by Fast FluoroAlkylation of Proteins (FFAP). FFAP is a novel protein labeling method relying on fluoroalkyl radicals generated from hypervalent Togni reagents. These reactive species target Cysteine, Histidine, Phenylalanine, Tryptophane and Tyrosine and are beneficial for structural characterization of proteins and protein complexes.

Methods

The mono- or bi-valent Togni reagents are decomposed by the ascorbic acid at the native environment to form a fluoroalkyl radicals that decorate the solvent accessible aromatic and sulfhydryl amino acid side chains of any protein at a short time period utilizing quench-flow microcapillary apparatus. Further, the protein samples are reduced, alkylated and digested by trypsin/Lys-C. Resulting peptides are separated on reversed-phase column (Luna Omega Polar C18, 100Å, Phenomenex) in water-acetonitrile gradient (Agilent 1290, Agilent Technologies) and directly analyzed by high resolution mass spectrometry (solariX XR 15T or timsTOF SCP, Bruker Daltonics). For mono-valent probe, the regular and modified peptides are identified by FragPipe (v.20.0) and the extent of modification is calculated from the intensity of the first isotope at the maximum of the chromatographic peak using DataAnalysis software (Bruker Daltonics). In case of bi-valent probe, the cross-linked peptides are fished out from the complex fragment spectra thanks to MeroX 2.0.

Results

First, the mono- and bi-valent probes shed a light on complex formation of human Hemoglobin-Haptoglobin complex. Second, mono-valent radicals were successfully tested for epitope mapping on the complex of Trastuzumab with Her2. The differences in the modification of free proteins and in the complex uncovered residues located on interaction interface that nicely correspond with previous structural analysis. Moreover, these data revealed a minor structural rearrangement of HER2 receptor upon Trastuzumab binding which is explained by remodeling of β‑sheet structure to unstructured loop. Similar behavior was already reported for rat Her2 (1N8Y) and human HER2 receptor – Pertuzumab complex. Further, the FFAP was subsequently used for the study of the structural differences between E. coli chloride channel (CLC-ec1) and its QQQ mutant. Results showed a critical connection between protonation and structural changes in the ion transport path and conformational change in the transmembrane helixes.