The Study On The Orientation Of Single-helix Transmembrane Peptides In Mimic Membrane

Correct orientation of transmembrane peptides and proteins in membrane is essential for them to obtain biological functions.However,the mechanism determining how they orientate is not yet clear.It is therefore very important to reveal the key factors controlling the orientation of transmembrane peptides and proteins in membrane,and to investigate their working mechanism.TAMRA,a fluorescent molecule with very high extinction coefficient,was applied as fluorescent probe and tryptophan as quencher to establish a new method to characterize the orientation of single-helix transmembrane peptides in membrane.The effects of various factors,including the sequence,the chain and the charge states of peptides,as well as the composition,the charge and the curvature of the mimic membrane,have been systematically investigated.In this thesis,we firstly designed 8 single-helix peptides N-terminally labelled with TAMRA and used extrusion method to obtain homogeneous lipid vesicles(POPC,POPG and DOPC),which are characterized by dynamic light scattering and transmission electron microscopy respectively to have a view of the size and morphology of lipid vesicles.Secondly,circular dichroism spectra and anisotropy are applied to verify the reconstruction degree of peptides in mimic membrane and the appropriate concentration ratio.Steady-state fluorescence and Time-resolved fluorescence techniques were applied to analyze the orientation distribution and influence factors.The orientation of transmembrane peptides in the membrane was determined based on the experimental data.Several important conclusions are obtained from fluorescence quencheing results.For one type of mimic membrane,the orientation distribution is almost the same,independent of the sequence and length of peptides.Ion concentration has no effect on the orientation distribution.The ratio for peptides in neutral membranes does not change if we reduce the pH value of solution,however the low pH has a significant effect on orientation distribution for the peptides in negatively charged membrane.This work provides important information for understanding the folding mechanism of membrane proteins.