Accessibility and mobility of lysine residues in .beta.-lactoglobulin

Abstract

N.epsilon.-[2H6]Isopropyllysyl-.beta.-lactoglobulin was prepared by reductive alkylation of .beta.-lactoglobulin with [2H6]acetone and NaBH4 to provide a 2H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein''s 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Lys47 was not modified; Lys135,138,141, located along an amphipathic helical rod, were each partially unmodified. All other lysine residues were at least 90% modified. Average correlation times calculated from 2H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. 2H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of .beta.-lactoglobulin previously reported from 31P MNR studies of the phospholipids complexed with .beta.-lactoglobulin. Although no physiological function has yet been identified, comparision of these results with X-ray structure [Papiz et al. (1986) Nature (London) 324, 383-385] supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped .beta.-barrel thought to contain binding sites for small lipid-soluble molecules.