Use of Gold Nanoparticles as Additives in Protein Crystallization Diana Ribeiro, † Alina Kulakova, † Pedro Quaresma, ‡ Eula ́ lia Pereira, ‡ Cecília Bonifa ́ cio, † Maria Joa ̃ o Roma ̃ o, † Ricardo Franco, †, * and Ana Luísa Carvalho †, * † REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciê ncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal ‡ REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciê ncias da Universidade do Porto, 4169-007 Porto, Portugal ABSTRACT: Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macro- molecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphol- ogy, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAH), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions. ■ INTRODUCTION X-ray crystallography is the foremost method to acquire atomic resolution for protein structures, and the limiting step is still the production of protein crystals suitable for structure solution. Therefore, strategies that facilitate the production of well- ordered crystals for X-ray diffraction techniques, such as nucleating agents and additives, are highly sought for. Gold occupies a unique position among the elements of the periodic table, and its chemical stability, the useful surface chemistry of the materials it generates, and its distinctive optical properties, have made gold an extremely attractive metal to employ in a variety of technologies. 1-3 This is especially true for nanotechnology as a consequence of these unique properties, as it is easier to work with gold at the nanoscale than with any other metal. 2,3 Gold nanoparticles (AuNPs) present several advantages, such as the control over size and morphology at the nanometer- scale, as well as the ability of functionalization with bioactive materials. 4 AuNPs have excellent biocompatibility and display unique structural, electronic, magnetic, optical, and catalytic properties, which have made them a very attractive material for the development of bionanosystems, 5 based on coupling AuNPs with biological macromolecules. Given its wide applications in biomaterials and interesting interactions, AuNPs could be potential agents for use in protein crystallization experiments. In 2008, Hodzhaoglu et al. have described AuNPs as effective nucleants for lysozyme (HEWL) crystallization. 6 Their study revealed an increase of the nucleation number of HEWL in the presence of citrate-capped AuNPs and of AuNPs functionalized with alkanethiols presenting a COOH terminal group. Their results suggested that AuNPs induced the crystallization of HEWL and also of ferritin. 6 These observations were further explored in the present study, in order to evaluate a possible application to other proteins less prone to crystallization, or to yield well-diffracting crystals. ■ METHODS AuNPs Synthesis and Functionalization. Spherical AuNPs were synthesized by the Turkevich method with minor modifications, 7,8 in which HAuCl 4 is chemically reduced by citrate (Table 1). A solution containing 62.5 mL of Milli-Q water and 43 μL of a 30 wt % gold salt solution was heated until boiling under reflux, using a sand-bath, with continuous stirring. At this point 6.25 mL of 36.8 mM sodium citrate solution was quickly added. The solution immediately changed color from the gold complex characteristic yellow color to colorless, then to black, and finally to red. Heating and stirring was continued for another 15 min, and the nanoparticle colloid was cooled down to room temperature. The method proposed by Haiss et al. 9 was used to estimate the concentration of AuNPs. The AuNP solution was stored at 4 °C until further use. The average size of citrate-capped AuNPs prepared following the same procedure and under the same conditions Received: September 23, 2013 Revised: November 7, 2013 Published: November 13, 2013 Article pubs.acs.org/crystal © 2013 American Chemical Society 222 dx.doi.org/10.1021/cg4014398 | Cryst. Growth Des. 2014, 14, 222-227