Solvent mimicry with methylene carbene to probe protein topography mimetic carbene reagent

G.E. GÓMEZ; MONTI, J.L; MUNDO, M.R.; DELFINO J.M

ANALYTICAL CHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2015 vol. 87 p. 10080 - 10087

0003-2700

The solvent accessible surfacearea (SASA) of the polypeptide chain plays a key role in protein folding,conformational change and interaction. This fundamental biophysical parameteris elusive in experimental measurement. Our approach to this problem relies onthe reaction of the minimal photochemical reagent diazirine (DZN) withpolypeptides. This reagent (i) exerts solvent mimicry because its size iscomparable to water and (ii) shows scant chemical selectivity because itgenerates extremely reactive methylene carbene. Methylation gives rise to theEM (extent of modification) signal, which is useful for scrutinizing theconformational change triggered by Ca2+ binding to calmodulin (CaM).The increased EM observed for the full protein is dominated by the enhancedexposure of hydrophobic area in Ca2+-CaM.Fragmentation allowed us to quantify the methylene incorporation at specificsites. Peptide 91-106 reveals a major reorganization around the calcium 151binding site, resulting in local ordering and a greater exposure of thehydrophobic surface. Additionally, this technique shows a high sensitivity toprobe recognition between CaM and melittin(Mel). The large decrease in EM indicates the occlusion of a significanthydrophobic area upon complexation. Protection from labeling reveals a largerinvolvement of the N-terminal and central regions of CaMin this interaction. Despite its smaller size, Mel?s differential exposure canalso be quantified. Moreover, MS/MS fragmentation realizes the goal ofextending the resolution of labeled sites at the amino acid level. Overall, DZN labeling emerges as a usefulfootprinting method capable of shedding light on physiological conformationalchanges and interactions.