IDENTIFYING THE LIPID-PROTEIN INTERFACE OF THE TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR - SECONDARY STRUCTURE IMPLICATIONS
IDENTIFYING THE LIPID-PROTEIN INTERFACE OF THE TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR - SECONDARY STRUCTURE IMPLICATIONS
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DOI：
10.1021/bi00176a016
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发表时间：
1994-03-15
期刊：
影响因子：
2.9
通讯作者：
COHEN, JB
中科院分区：
文献类型：
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作者：
BLANTON, MP;COHEN, JB
为了鉴定与膜脂相互作用的鱼雷烟碱乙酰胆碱受体（AchR）的氨基酸残基，我们使用了光活化的疏水探针3-三氟甲基-3-（m-[I-125]-碘苯基）重氮嘧啶（[I-125]TID）。[I-125]TID掺入每个亚基的M3和M4疏水段的模式在存在和不存在激动剂氨甲酰胆碱以及存在过量的非放射性TID时都是相同的，与脂质-蛋白界面的非特异性光掺入一致。[1 -125] [Blanton， M. P., Cohen， J. B.（1992）生物化学31,3738-3750]但只与β -、γ -和δ -亚基的M4片段上的2或3个残基反应。在delta-M3中，[I-125]TID与Met-293、Ser-297、Gly-301、Val-304和Asn-305以及M3之前的Ile-288发生反应。在β - m3 （Met-285、Ile-289、ph -293）和γ - m3 （ph -292、Leu-296、Met-299和Asn-300）以及γ - ile -283中标记相应位置的残基。在alpha-M3中，标记了ph -284和Ser-287。标记残基的周期性提供了第一个直接证据，证明每个亚基的M3和M4片段都组织为跨膜α -螺旋，每个都与脂质有大量接触。此外，在α - m1 [I-125]中，TID与Cys-222、Leu-223、Phe-227和Leu-228发生非特异性反应，这种结合模式与α -螺旋或β -片的“面”的标记模式不一致。
To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[I-125]-iodophenyl)diazirine ([I-125]TID). The pattern of [I-125]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [I-125]TID reacted with five residues in alpha-M4 [Blanton, M. P., and Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [I-125]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [I-125]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a ''face'' of an alpha-helix or a beta-sheet.