Protein Structure Dynamics and Function at Micelle and Particle Interfaces

Edición de la obra Protein Structure Dynamics and Function at Micelle and Particle Interfaces

This work is an investigation into how protein function and structural dynamics are affected by their interactions with detergents and particles. Recent studies have reported the addition of certain non-ionic surfactants increase the catalytic rates of enzymes. This is a departure from the established understanding of surfactant/protein interactions wherein charged-surfactants tend to denature the macro-molecular structure, while other detergents can be used to isolate and preserve protein structures. The mechanism of denaturation by ionic surfactants is well understood, as is the preservation and crystallization of protein structures with detergents. But why should non-ionic surfactants increase observed activity? This question remains unanswered. Furthermore, the explanations provided with previous studies where enzyme activity enhancement via surfactant interactions are inconsistent with each other, and are highly specific to the system in which they are studied. These contradicting conclusions highlight the need to propose a general theory of protein-surfactant interactions which can explain these reported changes in enzyme activity that can be applied to any of these reported systems. The theory proposed in this thesis is as follows: Unlike ionic surfactants, non-ionic surfactants minimally bind to the protein structure. Instead, bound water regions surrounding non-ionic surfactant aggregates (micelles) interact with the surface hydration of adjacent proteins, thus affecting the free energy of solvent forces at the protein surface. This, in turn, affects the small scale fluctuations of the proteins structures which influenced properties important to enzyme function, such as flexibility and substrate binding energy. Aside from enzyme interactions with surfactant, the implications of this proposed theory extend to any system in which functional proteins readily interact with a surface. The two parallel examples also reported in this dissertation are the structural dynamics of carrier proteins adsorbed onto metal phosphate adjuvant particles and a mathematical model describing changes to FRET efficiency due to the fluctuating motions of a light active protein. Three major objectives were established in order to test this hypothesis: 1) Establish model system of enzyme and surfactants, and a means to test enzyme efficacy. 2) Study the differences in surfactants and surfactant aggregates that would attribute to changes in enzyme activity and 3) Test the flexibility or the enzyme structures as a function of surfactant structure and concentration to see if there is, indeed, a connection between the surfactant and the enzyme activity through a modification of the protein's structural dynamics. Subtilisin proteinase and horseradish peroxidase were ultimately chosen as model enzymes to study. Initial activity assays confirmed previously reported results in which charged surfactants such as sodium dodecyl sulfonates had little effect or negatively impacted enzyme activity. Longer exposures to these anionic surfactants result in a loss of activity which can be attributed to enzyme denaturation. Enzyme activity in non-ionic surfactants such as Polyoxyethylene (4) lauryl ether (Brij-30) and alkyl polyglucosides (APG), increased. Curiously, dodecyl-β-D-maltoside (DM), a sugar-based non-ionic surfactant of similar head group structure to APG, did not induce enhanced enzyme activity, giving us two similar surfactants with which we can compare to determine what factors, if not surfactant molecular structure, affect enzyme activity. Surface tension, dynamic light scattering, electron spin resonance and NMR were used to elucidate the structural differences between alkyl polyglucoside (APG) and dodecyl maltoside micelles (DM), and which of these characteristics are attributing to the differences in activity. It was found that APG transitions between two micelle structure conformations as a function of concentration. After surpassing an initial critical micelle concentration, APG forms loosely packed aggregates that are attracted to each other to form even larger super-aggregates. Passing a second transitional concentration, the micelles form dense, rod-like structures. It should be noted that enzyme activity peaks in the presence of the loosely-packed super-aggregates and drops back to normal values once the micelles transition into their rod-like configuration. Conversely, dodecyl maltoside, which did not induce greater enzyme activity, does not exhibit this dual phase transition. Instead, the surfactants in DM solutions exceeding the critical micelle immediately form tightly packed, single layered spherical micelles. From these experiments we conclude that the densely packed micelles like those formed by DM and APG at higher concentrations do not affect enzyme structure the same way as those loosely packed ones at lower APG concentrations. Life-time fluorescence techniques were employed to fulfill the third objective