Ridder Anja N J A, Spelbrink Robin E J, Demmers Jeroen A A, Rijkers Dirk T S, Liskamp Rob M J, Brunner Josef, Heck Albert J R, de Kruijff Ben, Killian J Antoinette
Department of Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Biochemistry. 2004 Apr 20;43(15):4482-9. doi: 10.1021/bi049899d.
In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.
在本研究中，提出了一种新方法，通过该方法可以直接研究跨膜肽的分子环境。这是通过将光活化交联探针掺入模型跨膜肽的疏水片段中来实现的。当该肽掺入脂质双层并照射紫外光时，交联探针与脂质之间形成共价键。这种交联反应可以通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳可视化，所得产物可以通过质谱进行表征。通过使用磷脂酶，证明该肽与脂质的两条酰基链交联。该肽表现出明显倾向于分配到流动性脂质中，并被排除在凝胶相脂质之外。然而，当该肽掺入含有两种不同酰基链长度脂质种类的双层中时，未观察到基于疏水匹配的肽周围脂质的分子分选。有人提出，跨膜部分的大小在膜蛋白与周围脂质的动态相互作用中起着重要作用，因此在决定是否能发生分子分选方面也起着重要作用。
