Blanton M P, Cohen J B
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1994 Mar 15;33(10):2859-72. doi: 10.1021/bi00176a016.
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-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]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. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & 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, [125I]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 [125I]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.
为了鉴定与膜脂相互作用的电鳐烟碱型乙酰胆碱受体（AchR）的氨基酸残基，我们使用了可光活化的疏水探针3-三氟甲基-3-（间-[¹²⁵I]-碘苯基）重氮甲烷（[¹²⁵I]TID）。在有和没有激动剂氨甲酰胆碱的情况下以及在存在过量非放射性TID的情况下，[¹²⁵I]TID掺入每个亚基的M3和M4疏水片段的模式相同，这与从脂-蛋白界面的非特异性光掺入一致。[¹²⁵I]TID与α-M4中的五个残基发生反应[布兰顿，M.P.，& 科恩，J.B.（1992年）《生物化学》31卷，3738 - 3750页]，但与β-、γ-和δ-亚基的M4片段中的仅两个或三个残基发生反应。在δ-M3中，[¹²⁵I]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、Phe-293）和γ-M3（Phe-292、Leu-296、Met-299和Asn-300）以及γ-Ile-283中相应位置的残基被标记。标记残基的周期性提供了首个直接证据，即每个亚基的M3以及M4片段都被组织成跨膜α-螺旋，每个α-螺旋都与脂质有大量接触。此外，在α-M1中，[¹²⁵I]TID与Cys-222、Leu-223、Phe-227和Leu-228发生非特异性反应，这种掺入模式与α-螺旋的“面”或β-折叠预期的标记模式不一致。
