Structural Stability Effects on Adsorption of Bacteriophage T4 Lysozyme to Colloidal Silica

Abstract

Circular dichroism (CD) spectra were obtained for bacteriophage T4 lysozyme and three of its mutants in the presence and absence of colloidal silica nanoparticles. Mutant lysozymes were produced by substitution of the isoleucine at position 3 with tryptophan, cysteine, and leucine. Each substitution resulted in an altered structural stability, quantified 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 recorded after contact for 90 min showed significant differences from those recorded in the absence of nanoparticles, and these differences varied among the proteins. The percentage of α-helix lost in these proteins 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 adsorption. These kinetic data were evaluated using a model allowing proteins to exist in two different conformational states at the interface: state 1 molecules retain their native conformation, and state 2 molecules lose a certain amount of secondary structure and occupy more surface area than state 1 molecules. This analysis indicated that proteins of lower thermal stability have a greater tendency to adopt state 2 on silica. Rate constants governing generation of state 1 and state 2 molecules determined by CD were used as initial values of surface coverage-dependent rate constants in order to simulate adsorption kinetics. Comparison of simulated curves to adsorption data recorded within situellipsometry suggests that protein adsorption may adequately be described with a model allowing for only two functional states.