Aggregation state of spin-labeled cecropin AD in solution.
A spin-labeled derivative of the ion channel peptide cecropin AD (Fink et al., 1988) was synthesized and used to investigate its aggregation state in water and in the presence of a helix-promoting solvent. A cysteine was introduced at position 33 and spin-labeled using the methanethiosulfonate spin label. In low ionic strength aqueous solution, the peptide is monomeric, and the ESR spectrum indicates a high degree of segmental flexibility at the nitroxide attachment point, consistent with a predominantly random coil conformation. Upon addition of 5-10% (v/v) hexafluoro-2-propanol (HFP), the peptide is induced to aggregate as evidenced by significant motional restriction of the spin label and spin-spin broadening of the ESR lines. At higher concentrations of HFP, the peptide reverts to a monomeric state but retains its folded conformation. Our data suggest that between 5 and 10% HFP the peptide undergoes two structural transitions. The first transition starts at 5% and is very cooperative. Its dependence on ionic strength, temperature, and pH indicates that it involves the interconversion between a random coil and an ordered state stabilized by interpeptide electrostatic and hydrophobic interactions. The second transition, which occurs at 11% v/v HFP, is between the self-associated form and an ordered monomeric form. The analysis of our experimental results demonstrates aggregate formation at 5-10% HFP. This may be relevant to the mechanism of channel formation by cecropins in membranes.
Mchaourab
H S
HS
Biophysics Research Institute, Medical College of Wisconsin, Milwaukee 53226.
Hyde
J S
JS
Feix
J B
JB
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007202:Indicators and Reagents; D007301:Insect Hormones / Q000138:chemical synthesis / Q000737:chemistry*; D019476:Insect Proteins; D007473:Ion Channels / Q000737:chemistry; D008969:Molecular Sequence Data; D020005:Propanols; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012996:Solutions; D013113:Spin Labels; D013816:Thermodynamics; D014508:Urea; D006845:Hydrocarbons, Fluorinated
D003497:Cyclic N-Oxides; D003545:Cysteine; D007202:Indicators and Reagents; D007301:Insect Hormones; D019476:Insect Proteins; D007473:Ion Channels; D020005:Propanols; D012996:Solutions; D013113:Spin Labels; D014508:Urea; C084230:cecropin AD protein, insect; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; C003959:TEMPO