Lee Tzong-Hsien, Hall Kristopher N, Swann Marcus J, Popplewell Jonathan F, Unabia Sharon, Park Yoonkyung, Hahm Kyung-Soo, Aguilar Marie-Isabel
Department of Biochemistry and Molecular Biology, Monash University, Wellington Rd, Clayton, Vic, 3800, Australia.
Biochim Biophys Acta. 2010 Mar;1798(3):544-57. doi: 10.1016/j.bbamem.2010.01.014. Epub 2010 Jan 25.
The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.
使用两种互补的光学生物传感器对两种螺旋抗菌肽HPA3和HPA3P与平面支撑脂质膜的相互作用进行了定量分析。这些肽是源自幽门螺杆菌核糖体蛋白L1（RpL1）N端的Hp(2 - 20)类似物。利用表面等离子体共振（SPR）和双偏振干涉测量法（DPI）测定了这两种肽类似物与两性离子二肉豆蔻酰磷脂酰胆碱（DMPC）以及由DMPC/二肉豆蔻酰磷脂酰甘油（DMPG）（4:1）组成的带负电荷膜的结合情况。通过SPR分析表明，HPA3P中的脯氨酸取代导致其与两性离子膜和阴离子膜的结合比HPA3低得多。然后用DPI实时解析了两种Hp(2 - 20)类似物结合引起的平面DMPC和DMPC/DMPG（4:1）双层膜的结构变化。通过结合肽质量随双层膜双折射的实时变化分析了肽诱导膜结构变化的整个过程。HPA3和HPA3P插入支撑脂质双层都会导致双折射随着结合肽量的增加而降低，这反映了双层膜有序性的降低。与HPA3P相比，HPA3与每种膜的结合都伴随着更高水平的结合肽、更大程度的膜脂质无序以及更快且更高程度的插入膜中。此外，HPA3和HPA3P与带负电荷的DMPC/DMPG双层膜的结合也会导致脂质有序性受到更大破坏。这些结果证明了肽插入后膜的几何变化，并且可以定量获得膜结构变化的程度。此外，监测肽对具有结构特征的双层膜的影响，为膜结构变化在肽选择性和活性分子基础中的作用提供了进一步的见解，并可能有助于确定抗菌作用模式。
