Probing protein conformation with a minimal photochemical reagent

Abstract

H-diazirine (3 H-DZN), a photoreactive gas similar in size to water, was used to probe the topography of the surface and inner space of proteins. On photolysis 3 H-DZN generates 3 H-methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds. Labeling of bovine ␣-lactalbumin (␣-LA, MW: 14,200) with 1 mM 3 H-DZN yielded 0.0041 mol CH 2 /mol of protein, in agreement with the expectation for an unspecific surface-labeling phenomenon. The cooperative urea-induced unfolding of ␣-LA, as monitored by the extent of 3 H-methylene labeling, agrees with that measured by circular dichroism spectroscopy in the far and near ultraviolet regions. At 8 M urea, the unfolded state U was labeled 25-30% more than the native state N primarily because of the increase in the accessible surface area (ASA) of the protein occurring upon unfolding. However, this result lies below the ∼100% increment expected from theoretical estimates of ASA of state U. Among other factors, most likely the existence of a residual structure in U, that involves helices H2 and H4 of the ␣ subdomain, might account for this fact, as shown by a comparative analysis of peptide labeling patterns of N and U samples. In this paper, we demonstrate the usefulness of the 3 H-methylene labeling method to monitor conformational transitions and map solvent accessibility along the polypeptide sequence, thus opening the possibility of outlining structural features of nonnative states (i.e., denatured states, molten globule). We anticipate that this technique also would help to identify ligand binding and oligomerization sites in proteins.

Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

“…The build-up of methanol at low millimolar concentration poses no disturbance to protein conformation. Indeed, in previous work, samples subjected to diazirine reagent after irradiation were controlled by the very sensitive standard given by farand near-UV CD spectroscopy, showing identical spectra to non-treated samples [33,37]. This evidence rules out any putative change to conformation as a result of the presence of the diazirine reagent and the irradiation conditions.…”

“…In addition, to achieve the closest geometrical mimicry to the water solvent, the choice of methylene (:CH 2 ), the smallest and most reactive member of this family, emerges as a natural candidate [6,33]. To serve as a useful labeling reagent, :CH 2 has to be generated in situ from a suitable stable precursor.…”

“…Due to its extremely short half-life, : CH 2 will react with target groups belonging to its immediate molecular cage regardless of their chemical nature. At present the main proof of the indiscriminate reactivity of : CH 2 with proteins comes from the uniformity of the labeling yield observed in the unfolded state of bovine α-lactalbumin [33,36] and Bacillus licheniformis β-lactamase [37] at the level of tryptic and CNBr-derived peptides, in spite of differences in the amino acid composition of these peptides. This behavior and the minimal size of the precursor molecule represent key features of this experimental approach to probing the surface theoretically defined as SASA.…”

“…Most importantly, the modification with :CH 2 involves the formation of strong covalent bonds, giving rise to stable products that broaden the range of subsequent analytical procedures [6,33]. On the other hand, the extent of protein or water labeling results proportional to their relative abundance in the solution [33].…”

Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes

“…The simplest diazirine synthesized is unsubstituted and exists as a gas. Protein folding mechanisms and states have been probed using this simple photoreactive gas 53,54 . Aliphatic diazirine use is not as prevalent as the aromatic diazirines due to rearrangements resulting from the intermediate carbenes produced during photolysis often moving from the original site of generation.…”