Experimentally approaching the solvent accessible surface area of a protein. Insights into non-native states of bovine alpha lactalbumin

PATRICIO O. CRAIG; GABRIELA E. GÓMEZ; DANIELA B. URETA; JULIO J. CARAMELO; JOSÉ M. DELFINO

JOURNAL OF MOLECULAR BIOLOGY

Elsevier

Año: 2009 vol. 394 p. 982 - 993

0022-2836

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that in turn largely determines its stability. 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 the aqueous solvent. Here we structurally characterize non-native states of the paradigmatic protein a-lactalbumin (a-LA). Covalent tagging resulting from the unspecific methylene (:CH2) reaction allows one to obtain a global estimate of ASA and to map out the solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized (A) state of a-LA, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this A state also exhibits local regions where increased :CH2 labeling indicates its non-homogenous 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, :CH2 labeling emerges as a fruitful technique able to quantify the ASA of the polypeptide chain, thus probing conformational features like the outer exposed surface and inner cavities, as well as revealing the existence of non-compact apolar phases in non-native states.