Using gold nanoparticles in protein crystallography: studies in crystal growth and derivatization

Dissertation for the Master Degree in Structural and Functional Biochemistry

In the field of protein structural analysis, X-ray crystallography plays a major role to expand the knowledge on how proteins function, as well as their interactions with other molecules. However, several obstacles can be present when attempting to grow protein crystals and subsequently determine its structure. Therefore, strategies that facilitate the production of suitable crystals for X-ray diffraction techniques, such as nucleating agents, or that allow overcoming the phase problem, as the production of heavy atom derivatives, are highly sought for. Additionally, coupling gold nanoparticles (AuNPs) with proteins is of emerging interest and a field that has been growing in recent years, for the development of bionanosystems that benefit of the advantages presented by AuNPs. Understanding these processes at the atomic level, would facilitate the study of the interactions between proteins and AuNPs, and allow a deeper insight into these bionanosystems for their development and optimization. The number of studies reported in the literature combining protein crystallography and AuNPs is, up to date, very limited. The work developed in this Dissertation aimed to study the crystallization of proteins in the presence of AuNPs, associated to the study of its structural interactions with the AuNPs and gold atoms, and also to investigate the hypothesis of producing gold derivatives of protein crystals using AuNPs. To accomplish this, crystallization studies of HEWL (hen egg-white lysozyme) were carried out in the presence of gold nanoparticles capped with Polyvinylpyrrolidone (AuNP-PVP) and in the presence of a Au(I) compound. X-ray diffraction data from the resultant crystals were then measured at one of the X-ray absorption edges of gold, in order to take advantage of the gold‟s scattering properties. HEWL structures were then determined exhibiting several binding sites for gold atoms at the protein‟s surface that were unambiguously identified by recurring to an electron density map calculated from the anomalous differences of the measured intensities. In addition, TEM analyses were carried out to verify the assembly and formation of the AuNPs within HEWL crystals. Zeta-potential and optic spectroscopic measurements were also used to study the AuNP-PVP stability alone and in solution with HEWL. The potential of gold nanoparticles-induced crystal growth was also explored by using differently functionalised AuNPs as nucleant agents in co-crystallization studies with several proteins. Quite positive results were obtained with the test proteins, HEWL and RNase A (ribonuclease A), and a slight improvement of human phenylalanine hydroxylase (PAH) crystallization was also observed.