STRUCTURAL STABILITY EFFECTS ON ADSORPTION OF BACTERIOPHAGE-T4 LYSOZYME TO COLLOIDAL SILICA

Circular dichroism (CD) spectra were obtained for bacteriophage T4 lys ozyme and three of its mutants in the presence and absence of colloida l silica nanoparticles, Mutant lysozymes were produced by substitution of the isoleucine at position 3 with tryptophan, cysteine, and leucin e. Each substitution resulted in an altered structural stability, quan tified by a difference in free energy of unfolding from the wild type. CD spectra recorded in the absence of colloidal silica agreed with X- ray diffraction data in that the mutants and wild type showed similar secondary structures. CD spectra of protein-nanoparticle complexes rec orded after contact for 90 min showed significant differences from tho se recorded in the absence of nanoparticles, and these differences var ied among the proteins. The percentage of a-helix lost in these protei ns upon adsorption was also recorded as a function of time by CD, For a 1:2 protein to nanoparticle mixture, the more unstable the protein, the greater the rate and extent of secondary structure loss upon adsor ption, These kinetic data were evaluated using a model allowing protei ns to exist in two different conformational states at the interface: s tate 1 molecules retain their native conformation, and state 2 molecul es lose a certain amount of secondary structure and occupy more surfac e area than state 1 molecules. This analysis indicated that proteins o f lower thermal stability have a greater tendency to adopt state 2 on silica. Rate constants governing generation of state 1 and state 2 mol ecules determined by CD were used as initial values of surface coverag e-dependent rate constants in order to simulate adsorption kinetics. C omparison of simulated curves to adsorption data recorded with in situ ellipsometry suggests that protein adsorption may adequately be descr ibed with a model allowing for only two functional states. (C) 1998 Ac ademic Press.