Probing protein conformation with a minimal photochemical reagent.
3H-diazirine (3H-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 3H-DZN generates 3H-methylene carbene, which reacts unselectively with its molecular cage, inserting even into C-H bonds. Labeling of bovine alpha-lactalbumin (alpha-LA, MW: 14,200) with 1 mM (3)H-DZN yielded 0.0041 mol CH2/mol of protein, in agreement with the expectation for an unspecific surface-labeling phenomenon. The cooperative urea-induced unfolding of alpha-LA, as monitored by the extent of 3H-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 approximately 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 alpha 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 3H-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.
Craig
Patricio O
PO
Departamento de Química Biológica-IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Ureta
Daniela B
DB
Delfino
José M
JM
D000818:Animals; D002417:Cattle; D003978:Diazomethane / Q000528:radiation effects; D007768:Lactalbumin / Q000737:chemistry; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000528:radiation effects; D010782:Photolysis; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D011506:Proteins / Q000737:chemistry*; D014316:Tritium; D014508:Urea / Q000494:pharmacology
D015335:Molecular Probes; D011506:Proteins; D014316:Tritium; D003978:Diazomethane; D014508:Urea; D007768:Lactalbumin