Pervushin K V, Popov A I, Arseniev A S
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
Eur J Biochem. 1994 Jan 15;219(1-2):571-83. doi: 10.1111/j.1432-1033.1994.tb19973.x.
Spatial structures of a chymotryptic fragment C2 (residues 1-71) of bacterioopsin from Halobacterium halobium, solubilized in a mixture of methanol/chloroform (1:1, by vol.) and 0.1 M 2HCO2NH4, or in perdeuterated sodium (2H)dodecyl sulfate (SDS) micelles in the presence of perdeuterated (2,2,2-2H)trifluoroethanol, were determined by two-dimensional and three-dimensional heteronuclear 15N-1H NMR techniques. The influence of (2,2,2-2H)trifluoroethanol on the conformational dynamics of C2 in micelles and the effect of the salt (organic mixture) were studied. Under the best conditions, 1H and 15N resonances of 15N-uniformly enriched protein were assigned in both milieus by homonuclear two-dimensional NOE (NOESY) and two-dimensional total-correlated (TOCSY) spectra and heteronuclear three-dimensional NOESY-multiple-quantum-correlation (HMQC) and TOCSY-HMQC spectra. 651 (organic mixture) and 520 (micelles) interproton-distance constraints, derived from volumes of cross-peaks in two-dimensional NOESY and three-dimensional NOESY-HMQC spectra, along with deuterium exchange rates of amide groups measured in both milieus and 51 HN-C alpha H coupling constants obtained in the case of the organic mixture, were used in the construction of C2 spatial structures. Obtained structures are similar in both milieus and have two right-handed alpha-helical regions stretching from Pro8 to Met32 and Phe42 to Tyr64 (organic mixture), and from Pro8 to Met32 and Ala39 to Leu62 (micelles). In micelles, the second alpha helix is terminated by C-cap Gly63, adopting a conformation characteristic of a left-handed helix. Residues Gly65 to Thr67 from the turn of a right-handed helix. In the isotropic medium of the organic mixture, the C-terminal region of residues 65-71 lacks an ordered structure. Torsion angles chi 1 were unequivocally determined for 18 alpha-helical residues in both milieus. In the isotropic organic mixture and anisotropic micellar system, C2 remains a compact structure with a characteristic size of 3.0-3.5 nm. C2 seems to be present in at least two conformational states, packed and unpacked. Using NMR data, along with the electron cryomicroscopy model of bacteriorhodopsin [Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckman, E. & Downing, K. H. (1990) J. Mol. Biol. 213, 899-929], we suggested a model for the conformation of C2 in this putative close-packed state. However, no NOE contact between alpha helices was found in either milieu.
嗜盐菌视紫红质胰凝乳蛋白酶水解片段C2（第1至71位氨基酸残基）溶解于甲醇/氯仿（体积比1:1）和0.1 M甲酸铵的混合液中，或溶解于全氘代十二烷基硫酸钠（SDS）胶束及全氘代（2,2,2 - 2H）三氟乙醇存在的环境中，其空间结构通过二维和三维异核15N - 1H NMR技术测定。研究了（2,2,2 - 2H）三氟乙醇对C2在胶束中构象动力学的影响以及盐（有机混合物）的作用。在最佳条件下，通过同核二维NOE（NOESY）和二维全相关（TOCSY）谱以及异核三维NOESY - 多量子相关（HMQC）和TOCSY - HMQC谱，在两种环境中对15N均匀富集的蛋白质的1H和15N共振进行了归属。从二维NOESY和三维NOESY - HMQC谱中的交叉峰体积得出651个（有机混合物）和52个（胶束）质子间距离约束，以及在两种环境中测得的酰胺基团的氘交换率和在有机混合物情况下获得的51个HN - CαH耦合常数，用于构建C2的空间结构。在两种环境中获得的结构相似，具有两个右手α - 螺旋区域，在有机混合物中从Pro8延伸至Met32和Phe42至Tyr64，在胶束中从Pro8延伸至Met32和Ala39至Leu62。在胶束中，第二个α - 螺旋由C - 帽Gly63终止，呈现左手螺旋的特征构象。右手螺旋转角处的残基Gly65至Thr67。在有机混合物的各向同性介质中，65 - 71位残基的C末端区域缺乏有序结构。在两种环境中，明确测定了18个α - 螺旋残基的扭转角χ1。在各向同性有机混合物和各向异性胶束系统中，C2保持紧凑结构，特征尺寸为3.0 - 3.5 nm。C2似乎至少以两种构象状态存在，紧密堆积和未堆积状态。利用NMR数据，结合细菌视紫红质的电子冷冻显微镜模型[亨德森，R.，鲍德温，J. M.，切斯卡，T. A.，泽姆林，F.，贝克曼，E. & 唐宁，K. H.（1990）《分子生物学杂志》213，899 - 929]，我们提出了C2在这种假定的紧密堆积状态下的构象模型。然而，在任何一种环境中都未发现α - 螺旋之间的NOE接触。
