Jones D H, Barber K R, VanDerLoo E W, Grant C W
Department of Biochemistry, University of Western Ontario, London, Canada.
Biochemistry. 1998 Nov 24;37(47):16780-7. doi: 10.1021/bi981520y.
As part of a study of receptor tyrosine kinase behavior in membranes, we have collected extensive NMR data from three well-defined probe locations within the transmembrane region of the human EGF receptor. Spectra were obtained for selectively deuterated alanine residues in a series of peptides corresponding to the putative transmembrane domain (with short extramembranous extensions). Peptides were incorporated into fluid unsonicated liposomes of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and POPC containing 33 mol % cholesterol to mimic common lipid composition of cell plasma membranes. The peptide concentration was in the range of 1-6 mol % relative to that of phospholipid. Data acquired at 35 degreesC have been analyzed quantitatively to determine their implications to receptor spatial orientation and dynamics. If it is presumed that the single transmembrane portion approximates an alpha-helix of 3.6 residues per turn, this helix was found to be tilted away from the membrane perpendicular, about which there was rapid axial diffusion. However, rotation about the peptide long axis was static on the NMR time scale of 10(-)4 s, and the peptide appeared to have a preferred direction(s) of lean. The results for this peptide, whose hydrophobic length is greater than the membrane hydrophobic thickness, were very similar between membranes of POPC and membranes of POPC containing 33 mol % cholesterol, despite considerable host matrix differences in thickness and order. Allowed values of peptide tilt occupied a narrow range: between 10 and 14 degrees in POPC and between 10 and 12 degrees in POPC/cholesterol. Although the existence of some preferred direction(s) of lean was demanded by the results, the direction of lean was not uniquely determined. We have interpreted these results, which were essentially unchanged at 65 degreesC, as reflecting the behavior of peptide monomers undergoing rapidly reversible peptide-peptide interactions. For transmembrane monomers, interference with rotation about the peptide long axis might be understood to arise from an energy benefit (in a tilted peptide) to prevention of particular amino acid side chains near the membrane surfaces from moving in and out of hydrophobic or hydrophilic environments. It will be desirable to test the conclusion of preferential lean of a monomeric receptor since such behavior could provide a mechanism for modulating monomer association with other species (i.e., signal transduction).
作为一项关于膜中受体酪氨酸激酶行为研究的一部分，我们从人表皮生长因子受体跨膜区域内三个明确的探针位置收集了大量核磁共振（NMR）数据。我们获得了一系列与假定跨膜结构域（带有短的膜外延伸部分）相对应的肽段中选择性氘代丙氨酸残基的光谱。这些肽段被掺入到由1 - 棕榈酰 - 2 - 油酰磷脂酰胆碱（POPC）以及含有33摩尔%胆固醇的POPC组成的流体且未超声处理的脂质体中，以模拟细胞质膜的常见脂质组成。相对于磷脂，肽段浓度在1 - 6摩尔%的范围内。对在35摄氏度下获取的数据进行了定量分析，以确定其对受体空间取向和动力学的影响。如果假定单个跨膜部分近似于每圈3.6个残基的α螺旋，那么发现该螺旋偏离膜垂直方向，并且存在快速的轴向扩散。然而，在10^(-4)秒的NMR时间尺度上，围绕肽段长轴的旋转是静态的，并且肽段似乎有一个优先的倾斜方向。对于这种疏水长度大于膜疏水厚度的肽段，尽管POPC膜和含有33摩尔%胆固醇的POPC膜在厚度和有序度方面存在相当大的宿主基质差异，但其结果非常相似。肽段倾斜的允许值占据一个狭窄范围：在POPC中为10至14度，在POPC/胆固醇中为10至12度。尽管结果表明存在一些优先的倾斜方向，但倾斜方向并未唯一确定。我们将这些在65摄氏度时基本不变的结果解释为反映了经历快速可逆肽 - 肽相互作用的肽单体的行为。对于跨膜单体，围绕肽段长轴旋转的干扰可能被理解为源于一种能量益处（在倾斜的肽中），即防止膜表面附近的特定氨基酸侧链进出疏水或亲水环境。检验单体受体优先倾斜的结论将是很有必要的，因为这种行为可能提供一种调节单体与其他物种结合（即信号转导）的机制。
