Girvin M E, Fillingame R H
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706.
Biochemistry. 1995 Feb 7;34(5):1635-45. doi: 10.1021/bi00005a020.
Purified subunit c from the H(+)-transporting F1F0 ATP synthase of Escherichia coli folds as an antiparallel pair of extended helices in a solution of chloroform-methanol-water. A similar hairpin-like folding is predicted for the native protein in the multisubunit transmembrane Fo sector of the ATP synthase. A single Cys variant (A67C) of subunit c was created and modified with a maleimido-PROXYL [[3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyl]oxy] spin label. Pairs of 1H 2D correlation and NOE spectra were collected with the nitroxide oxidized (paramagnetic) and reduced (diamagnetic). The pairs of spectra were subtracted, yielding difference spectra containing only cross-peaks from 1H within 15 A of the spin label. These greatly simplified spectra were easily analyzed to provide complete assignments for residues 10-25 and 52-79 of the protein, 150 NOE distance restraints, and 27 hydrogen-bonding restraints. The chemical shifts and NOE patterns observed in the derivatized mutant were virtually identical to those which were resolved in the unmodified wild-type protein, strongly suggesting that the spin label was not perturbing the protein structure. The restaints enabled us to calculate a detailed structure for this region of subunit c. The structure consisted of two gently curved helices, crossing at a slight (30 degrees) angle. The C-terminal helix was disrupted from Val60 to Ala62 near the essential Pro64. Asp61, the residue thought to undergo protonation--deprotonation with each H+ transported across the membrane, was in ver der Waals contact with Ala24. The proximity of these residues had been predicted from mutant analyses, where H+ translocation was retained on moving the Asp from position 61 to 24.
从大肠杆菌的H⁺转运F₁F₀ ATP合酶中纯化得到的亚基c，在氯仿 - 甲醇 - 水的溶液中折叠成一对反平行的延伸螺旋。对于ATP合酶多亚基跨膜F₀部分中的天然蛋白质，预计会有类似发夹状的折叠。构建了亚基c的单个半胱氨酸变体（A67C），并用马来酰亚胺 - PROXYL [[3 - （马来酰亚胺甲基） - 2,2,5,5 - 四甲基 - 1 - 吡咯烷基]氧基]自旋标记进行修饰。在氮氧化物氧化（顺磁性）和还原（抗磁性）状态下收集了1H二维相关谱和NOE谱对。将这些谱对相减，得到仅包含自旋标记15 Å范围内1H交叉峰的差谱。这些大大简化的谱很容易分析，从而为蛋白质的10 - 25位和52 - 79位残基提供了完整的归属、150个NOE距离限制和27个氢键限制。在衍生化突变体中观察到的化学位移和NOE模式与在未修饰的野生型蛋白质中解析的几乎相同，这强烈表明自旋标记没有干扰蛋白质结构。这些限制使我们能够计算出亚基c这一区域的详细结构。该结构由两个轻微弯曲的螺旋组成，以轻微（30度）的角度交叉。C端螺旋在关键的Pro64附近从Val60到Ala62中断。Asp61被认为在每个H⁺跨膜转运时会发生质子化 - 去质子化，它与Ala24处于范德华接触。这些残基的接近性已从突变分析中预测到，在将Asp从61位移到24位时仍保留了H⁺转运。
