Measurement of the secondary structure of adsorbed protein by circular dichroism. 1. Measurements of the helix content of adsorbed melittin.
A new circular dichroism (CD) technique is presented which quantifies, in situ, the changes in protein and peptide secondary structure upon adsorption at the quartz/liquid interface. Far-UV CD spectra of adsorbed proteins were recorded from several quartz interfaces contained in a specially constructed cell. Adsorbed, oriented alpha-helical spectra were recorded from hydrophilic and hydrophobic quartz using the bee venom peptide, melittin, which can be induced into an alpha-helical, tetrameric conformation in solution. The hydrophobic quartz provides a model system for oil-in-water emulsions and cell membranes. Surface concentrations were determined by radio-counting and were dependent on the nature of the surface. The characterization of these spectra has been partly achieved using far-UV CD spectra obtained from melittin adsorbed onto hydrophilic colloidal silica particles, where orientation effects are eliminated. Analysis of these spectra reveals considerable denaturation of the helical structures upon adsorption. Surface concentrations from the silica were determined from adsorption isotherms. The surface orientation of adsorbed melittin was dependent on the state of aggregation and hence degree of helicity of the molecule. These results support a model for the mode of action of melittin in lysing membranes.
Smith
L J
LJ
AFRC Institute of Food Research, Colney, Norwich UK.
Clark
D C
DC
D000327:Adsorption; D002942:Circular Dichroism; D008555:Melitten / Q000737:chemistry*; D008962:Models, Theoretical; D011487:Protein Conformation; D012822:Silicon Dioxide; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D008555:Melitten; D012822:Silicon Dioxide