Peptide-membrane binding is vital for many biological events,including the bacteria combating by antimicrobial peptides.Using the p H sensitive LAH4 peptide as model,we employed a convenient electron paramagnetic reso...Peptide-membrane binding is vital for many biological events,including the bacteria combating by antimicrobial peptides.Using the p H sensitive LAH4 peptide as model,we employed a convenient electron paramagnetic resonance(EPR)method to study the peptide-membrane binding process in artificial phospholipid membranes.Based on spectral changes of the nitroxide radical labeled to the peptides,we characterized binding kinetics and affinity of peptides to different phospholipid membranes.The binding affinity of LAH4 towards POPG was more than an order of magnitude higher than those towards DMPC and POPC.The binding kinetics showed that LAH4 initially bound to POPG much more quickly than to DMPC and POPC.Additionally,p H also affected the binding kinetics in LAH4-membrane interactions,which helped explain the p H dependent antimicrobial activity of LAH4.The method might be further used to monitor the membrane binding/cell penetration of antimicrobial peptide in living cells.展开更多
Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one...Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2.RDH8 is a member of the short-chain dehydrogenase/reductase family.Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathicα-helix and of 1 to 3 acyl groups at positions 299,302 and 3043.The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.Methods:To evaluate the membrane binding of RDH8,the full-length protein(aa 1-312),a truncated form(aa 1-296),its C-terminal segment(aa 281-312 and 297-312)as well as different additional variants of this segment were used.The truncated protein binds membranes less efficiently than the full-length form.Thus,the C-terminal segment of RDH8 is essential for the binding and has thus been further examined.The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F,W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment.Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(POPS)were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.Results:An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles.These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp=1.1 E6 to 1.7 E6.It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer.The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition ofα-helical,β-turn and unordered structures.Conclusions:The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles.These strength of binding is independent of the type of lipid and the presence of a mutation.展开更多
基金Seed Fund of Peking University(Grant No.BMU2018MC003)Peking University Health Science Center(Grant No.BMU20160566)+1 种基金the National Key Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China(Grant No.2016YFA0501203)the National Natural Science Foundation of China(Grant No.31470727,21874004)
文摘Peptide-membrane binding is vital for many biological events,including the bacteria combating by antimicrobial peptides.Using the p H sensitive LAH4 peptide as model,we employed a convenient electron paramagnetic resonance(EPR)method to study the peptide-membrane binding process in artificial phospholipid membranes.Based on spectral changes of the nitroxide radical labeled to the peptides,we characterized binding kinetics and affinity of peptides to different phospholipid membranes.The binding affinity of LAH4 towards POPG was more than an order of magnitude higher than those towards DMPC and POPC.The binding kinetics showed that LAH4 initially bound to POPG much more quickly than to DMPC and POPC.Additionally,p H also affected the binding kinetics in LAH4-membrane interactions,which helped explain the p H dependent antimicrobial activity of LAH4.The method might be further used to monitor the membrane binding/cell penetration of antimicrobial peptide in living cells.
文摘Background:Retinol dehydrogenase 8(RDH8)is a 312-amino acid(aa)protein involved in the visual cycle.Bound to the outer segment disk membranes of photoreceptors,it reduces all-trans-retinal to all-trans-retinol1 as one of the rate-limiting steps of the visual cycle2.RDH8 is a member of the short-chain dehydrogenase/reductase family.Its C-terminal segment allows its membrane-anchoring through the postulated presence of an amphipathicα-helix and of 1 to 3 acyl groups at positions 299,302 and 3043.The secondary structure and membrane binding characteristics of RDH8 and its C-terminal segment have not yet been described.Methods:To evaluate the membrane binding of RDH8,the full-length protein(aa 1-312),a truncated form(aa 1-296),its C-terminal segment(aa 281-312 and 297-312)as well as different additional variants of this segment were used.The truncated protein binds membranes less efficiently than the full-length form.Thus,the C-terminal segment of RDH8 is essential for the binding and has thus been further examined.The intrinsic fluorescence of tryptophan residues at positions 289 and 310 of the wild-type C-terminal segment of RDH8 and the mutants W289F,W310F and W310R have thus been used to determine their extent of binding to lipid vesicles and to monitor their local environment.Unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)or a mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(POPS)were used to mimic the phospholipid content of the outer segment disk membranes of photoreceptors.Results:An increase in fluorescence intensity and in fluorescence lifetime is observed upon increasing the concentration of lipid vesicles.These data allowed calculating values of partition coefficient of the C-terminal segment of RDH8 varying between Kp=1.1 E6 to 1.7 E6.It is noteworthy that the observation of a more intense shift to lower wavelengths upon membrane binding of the mutant W310R and W310F indicates a deeper incorporation of the remaining tryptophan residue at position 289 into the lipid bilayer.The secondary structure of the C-terminal segment of RDH8 observed by circular dichroism and infrared spectroscopy shows a superposition ofα-helical,β-turn and unordered structures.Conclusions:The peptides derived from the C-terminal segment of RDH8 show a strong binding to lipid vesicles.These strength of binding is independent of the type of lipid and the presence of a mutation.
