Assignment of amide 1H and 15N NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer.
Assignment of amide 1H and 15N NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer.
复制标题
洗涤剂溶解的 M13 外壳蛋白中酰胺 1H 和 15N NMR 共振的归属：外壳蛋白二聚体的模型。
作者：
G. D. Henry;B. Sykes
丝状噬菌体M13的主要外壳蛋白是一种50个残基的完整膜蛋白。洗涤剂增溶的M13外壳蛋白是一种很有前途的核磁共振结构确定的候选蛋白，因为该蛋白可以大量制备，而且含有蛋白质的胶束相当小。在我们的实验条件下，与十二烷基硫酸钠结合的外壳蛋白以二聚体形式存在，表观分子量为27,000。宽阔的谱线和较差的1H谱分辨率导致我们采用了15N定向的方法，其中外壳蛋白既均匀地标记了15N，又选择性地标记了[α-15N]丙氨酸、-甘氨酸、-缬氨酸、-亮氨酸、-异亮氨酸、苯丙氨酸、-赖氨酸、-酪氨酸和-蛋氨酸。使用羧肽酶消化、双标记和从相邻的指定的13C标记的羰基碳确定的酰胺质子交换率的独立知识，尽可能地指定了氮的共振峰。均一和选择性标记蛋白质的1H/15N异核多量子相干(HMQC)谱随后将酰胺氮与酰胺质子化学位移关联起来，并依次从同核NOESY和HMQC-NOESY谱完成指认。在从甲硫氨酸-28到苯丙氨酸-42的连续伸展中，显示出最慢的酰胺质子交换。该序列包括疏水核心的大部分共振，尽管它向蛋白质的C末端移动。强烈的NH到NH(i，i+1)核Overhauser增强是外壳蛋白的一个特征，它似乎主要是螺旋的。在20~25个残基之间产生2个并列的共振，这在均匀的15N标记的外壳蛋白的HMQC谱中可以清楚地看到。这些残基集中在从跨膜序列的开始延伸到C末端附近的无序区域的区域。我们认为，十二烷基硫酸酯结合的M13外壳蛋白由两个独立的结构域组成，一个是处于中等快速动态流动状态的N端螺旋，另一个是与第二单体发生广泛相互作用的长的、稳定的C末端跨膜螺旋。酰胺1H化学位移与这幅图一致；此外，在分子的C末端观察到明显的周期性。
The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.