Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.
Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is either the bulk solution or water within parallel or antiparallel transmembrane arrays of reconstituted alamethicin.
Dempsey
C E
CE
Department of Biochemistry, Bristol University, Avon, UK. dempsey@bsa.bristol.ac.uk
Handcock
L J
LJ
D000408:Alamethicin / Q000737:chemistry*; D000577:Amides / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry; D004355:Drug Stability; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D010713:Phosphatidylcholines; D014867:Water
D000577:Amides; D000900:Anti-Bacterial Agents; D008081:Liposomes; D010713:Phosphatidylcholines; D014867:Water; D000408:Alamethicin; D006859:Hydrogen; D003903:Deuterium; C017251:1,2-oleoylphosphatidylcholine