Accessibility and mobility of lysine residues in beta-lactoglobulin.
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 NMR studies of the phospholipids complexed with beta-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the 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.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D007782:Lactoglobulins; D008081:Liposomes; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009038:Motion; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D011487:Protein Conformation
D007782:Lactoglobulins; D008081:Liposomes; D010446:Peptide Fragments; D008239:Lysine