Assessing Native and Non-native Conformational States of a Protein by Methylene Carbene Labeling:  The Case ofBacillus licheniformisβ-Lactamase

Abstract

Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis beta-lactamase (BL-betaL) with 1 mM 3H-diazirine yielded 8.3 x 10(-3) mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary structure, a statement supported by the fact that the largest change occurs along the N-I portion of the N-I-U transition and along the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N, an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentation of labeled BL-betaL into peptides provides a sequential map of solvent accessibility. Thus, amino acid residues pertaining to the Omega-loop and to helices alpha5 and alpha6 line the major cavity of the protein, that is big enough to lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique) experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possible a comparative structural characterization of the native as well as non-native states.

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.…”

“…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.…”

“…This evidence rules out any putative change to conformation as a result of the presence of the diazirine reagent and the irradiation conditions. An added advantage of the technique is the fact that the rate of diazirine photolysis in aqueous solution remains invariable under diverse conditions of pH, ionic strength, or concentration of chaotropes commonly used [33,36,37].…”

“…3 Hdiazirine has been successfully employed to address unfolding transitions of single-domain proteins [33,36,37] and the contact surface of an antigen-antibody complex [38]. Other researchers took advantage of diazirine labeling to identify an alternative conformation of replication protein A [39].…”

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

“…More recently, diazirine precursors have been incorporated into cross-linkers [27,28] and covalent labeling agents [23,29,30]. BCarbene footprinting^has been successfully demonstrated using gaseous diazirine [30], simple aliphatic diazirines [23,31,32], and most recently aromatic diazirine derivatives [29]. Carbene labeling appears to offer the hallmarks of an excellent, adaptable labeling tool.…”