Experimentally approaching the solvent-accessible surface area of a protein: insights into the acid molten globule of bovine alpha-lactalbumin.
Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein alpha-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH(2)) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of alpha-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH(2) labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH(2) labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.
Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires e Instituto de Química y Fisicoquímica Biológica (Consejo Nacional de Investigaciones Científicas y Técnicas), Junín 956, C1113AAD Buenos Aires, Argentina.
Patricio O
PO
Craig
Gabriela E
GE
Gómez
Daniela B
DB
Ureta
Julio J
JJ
Caramelo
José M
JM
Delfino
D000818:Animals; D002417:Cattle; D003978:Diazomethane / Q000378:metabolism; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods
D012997:Solvents; D003978:Diazomethane; D007768:Lactalbumin