Solvent Mimicry with Methylene Carbene to Probe Protein Topography

Abstract

The solvent accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding, conformational change, and interaction. This fundamental biophysical parameter is elusive in experimental measurement. Our approach to this problem relies on the reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides. This reagent (i) exerts solvent mimicry because its size is comparable to water and (ii) shows scant chemical selectivity because it generates extremely reactive methylene carbene. Methylation gives rise to the EM (extent of modification) signal, which is useful for scrutinizing the conformational change triggered by Ca(2+) binding to calmodulin (CaM). The increased EM observed for the full protein is dominated by the enhanced exposure of hydrophobic area in Ca(2+)-CaM. Fragmentation allowed us to quantify the methylene incorporation at specific sites. Peptide 91-106 reveals a major reorganization around the calcium 151 binding site, resulting in local ordering and a greater exposure of the hydrophobic surface. Additionally, this technique shows a high sensitivity to probe recognition between CaM and melittin (Mel). The large decrease in EM indicates the occlusion of a significant hydrophobic area upon complexation. Protection from labeling reveals a larger involvement of the N-terminal and central regions of CaM in this interaction. Despite its smaller size, Mel's differential exposure can also be quantified. Moreover, MS/MS fragmentation realizes the goal of extending the resolution of labeled sites at the amino acid level. Overall, DZN labeling emerges as a useful footprinting method capable of shedding light on physiological conformational changes and interactions.

Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions

“…Diazirine-based footprinting agents, in contrast, offer the potential to use near-ultraviolet wavelengths (≈350 nm)–outside the absorbance window of amino acids–to generate highly reactive carbenes, which rapidly label the surface of a protein2021. It should be noted that this generic strategy is quite distinct from that of photoaffinity labelling, where a specific photoactive ligand is employed, and which therefore requires a different probe for each system studied22.…”

Flash Oxidation (FOX) System: A Novel Laser-Free Fast Photochemical Oxidation Protein Footprinting Platform

“…Additionally, the broadband UV output and high intensity of the custom high-pressure Xe flash lamp can be used to enable flash photochemistry other than FPOP. For example, photoinitiated carbene-based footprinting − could be initiated using the FOX system, with highly controllable reaction times. Similarly, the FOX system could be used for photoaffinity labeling or photo-cross-linking, offering broad UV irradiation potential and controllable irradiation parameters.…”

PepFoot: A Software Package for Semiautomated Processing of Protein Footprinting Data

“…Carbene footprinting is an alternative to the hydroxyl radical approach. It utilizes a highly reactive carbene generated in situ , typically from a diazirine reagent irradiated with a near-ultraviolet laser. − The rate of this insertion is on the order of nanoseconds, much faster than secondary structure folding events, making carbenes an attractive reactive intermediate for protein study. While initial studies used diazirine gas to generate methylene, more recent work has employed trifluoromethylaryldiazirines due to their stability, solubility, and efficacy. ,− …”