Department of Chemistry and Biochemistry, University of Arkansas, 119 Chemistry Building, Fayetteville, AR, 72701, USA.
Present address: Department Department of Neurobiology, Duke University Medical Center, 311 Research Drive, Durham, NC, 27710, USA.
Chembiochem. 2019 Nov 4;20(21):2784-2792. doi: 10.1002/cbic.201900282. Epub 2019 Sep 18.
Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW LAL LALALAL ALW LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific H-labeled alanine residues within the central sequence for detection by solid-state H NMR spectroscopy. The resulting pattern of [ H]Ala quadrupolar splitting (Δν ) magnitudes indicates the core helix for R GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R GWALP23 helix adopts primarily a surface orientation. The inclusion of 10-20 mol % cholesterol in DOPC bilayers drives more of the R GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.
膜蛋白对于许多细胞过程至关重要，但比可溶性蛋白更难研究。带电残基通常对膜蛋白功能有重要贡献。模型肽，如 GWALP23（乙酰-GGALW LAL LALALAL ALW LAGA-酰胺），可用于表征特定残基对跨膜蛋白结构域的影响。我们已将 GWALP23 中的 R8 和 R16 替换为 L8 和 L16，它们与肽中心等距，并在中央序列中掺入特定的 H 标记丙氨酸残基，以便通过固态 H NMR 光谱检测。所得 [H]Ala 四极分裂（Δν）幅度模式表明，R GWALP23 的核心螺旋明显倾斜，在具有饱和 C12:0 或 C14:0 酰基链（1,2-二棕榈酰-sn-甘油-3-磷酸胆碱（DLPC）或 1,2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱（DMPC））或不饱和 C16:1 Δ9 cis 酰基链的较薄双层中具有相似的跨膜取向。在 1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC；C18:1 Δ9 cis）双层中，表明存在多种取向，而在较长的、不饱和的 1,2-二二十碳烯酰基-sn-甘油-3-磷酸胆碱（DEiPC；C20:1 Δ11 cis）双层中，R GWALP23 螺旋主要采用表面取向。在 DOPC 双层中加入 10-20 mol%胆固醇会促使更多的 R GWALP23 螺旋种群向膜表面移动，从而使两个带电荷的精氨酸都能接触到界面脂质头部基团。结果表明，对于脂膜中精氨酸肽的动力学和螺旋取向，疏水性厚度和胆固醇含量比脂质饱和度更为重要。
