ACCESSIBILITY AND MOBILITY OF LYSINE RESIDUES IN BETA-LACTOGLOBULIN
ACCESSIBILITY AND MOBILITY OF LYSINE RESIDUES IN BETA-LACTOGLOBULIN
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DOI：
10.1021/bi00415a031
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发表时间：
1988-07-26
期刊：
影响因子：
2.9
通讯作者：
GREENBERG, R
中科院分区：
文献类型：
--
作者：
BROWN, EM;PFEFFER, PE;GREENBERG, R
N ε- [2 H6]异丙基赖氨酰-β-乳球蛋白是通过β-乳球蛋白与[2 H6]丙酮和NaBH 4反应，为研究赖氨酸参与脂质-蛋白质相互作用提供2 H（NMR）探针。氨基酸分析表明，蛋白质的15个赖氨酸残基的80%被标记。未修饰的赖氨酸残基通过CNBr，胰蛋白酶，胰凝乳蛋白酶的标记蛋白质的肽图。Lys 47未被修饰; Lys 135、138、141，位于沿着两亲性螺旋杆，各自是部分未修饰的。所有其他赖氨酸残基至少90%被修饰。在6 M盐酸胍中，由2 H NMR谱计算的8.7和3.3个残基的平均相关时间分别为20和320 ps;在非变性溶液中，6.5和3.2个残基分别获得70和320 ps的值，其余2.3个修饰的残基未观察到，表明无序或柔性区域中的赖氨酸残基侧链比稳定周期结构中的赖氨酸残基侧链更移动的。与二棕榈酰磷脂酰胆碱复合的蛋白质的2 H NMR光谱证实了β-磷脂酰胆碱的外膜蛋白类型行为。之前从与β-复合的磷脂的31 P MNR研究中报道的乳球蛋白乳球蛋白尽管尚未鉴定出生理功能，但这些结果与X射线结构的比较[Papiz等人（1986）Nature（伦敦）324，383-385]支持了这样的假设，即不能接近修饰的残基可能有助于稳定锥形β-葡聚糖酶。桶被认为含有小的脂溶性分子的结合位点。
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.