X-Ray Study of Herbaspirilum Seropedicae GlnB Protein Adsorbed on Silicon Adriana Freire Lubambo, 1 Elaine Machado Benelli, 2 Irineu Mazzaro, 1 Fabiano Yokaichyia, 3 Carlos Giles, 4 Paulo Ce´sar de Camargo * 1 Summary: GlnB-Hs protein is a globular member of PII-like family involved in the nitrogen control fixation in diazothoph organisms like H. seropedicae. In this work, GlnB-Hs is deposited by spin-coating on Si (111) and its self-assembling is investigated by AFM (atomic force microscopy) and X-ray at grazing incidence angles. GlnB-Hs forms circular 100 nm to 150 nm wide face-up donut shaped protein aggregates on silicon. Reflectivity and diffraction profiles from the protein deposits on Si(111) and Si(100), suggest ordered domains that are stable under the X-ray beam and have d-spacing compatible with protein dimensions already determined by classical protein crystallography. Keywords: adsorption; atomic force microscopy (AFM); biopolymer; globular protein GlnB-Hs; self-assembly; X-ray Introduction In the last few years, the study of protein adsorption at various interfaces has shown an increasing interest. The driven forces that rule protein adsorption have been investigated by numerous techniques because of the important and wide number of applications that range from biocompat- ibility, biosensor design, to ultra filtration membranes. [1,2] One appealing interest is the use of nanobiofilm templates for protein crystallization and protein structure determination. [3,4] The knowledge of protein structure has biochemical and biotechnological motiva- tions: Structure is related to protein func- tion, which means the ability of the protein to perform one special task, and the knowledge of protein function, leading us directly to the understanding of biological processes. So far, protein crystallization is a difficult and laborious task: it depends on finding the ideal conditions of the solution, which will allow crystallization and the results, are often unpredictable and irre- producible, resulting on crystals that have quality and dimensions often inappropriate to structural X-ray studies. When the crystal is formed, it is fragile due to weak interactions between proteins in the crystal lattice and has to be embedded in solvent solution to avoid cracking. [5] Furthermore, there are proteins reluctant to crystallize, for example, the knowing difficulty to prepare 3D crystal of membrane protein. [6] X-ray surface methods on the study of organic monolayers and lipid monolayers on liquid and solid interfaces have demon- strated its capability to extract information of structure and ordering. [7–11] Also, this technique was successfully applied for structure determination of a single protein layer on liquid interface. [12] The investiga- tion of protein thin film adsorbed on solid surface demands the use of strong radiation sources due to the small amount of material on the substrate (protein coverage of a few monolayers). The use of grazing incidence Macromol. Symp. 2006, 245–246, 140–146 DOI: 10.1002/masy.200651319 140 1 Department of Physics, Universidade Federal do Parana ´ , Curitiba, Brazil E-mail: camargo@fisica.ufpr.br 2 Department of Biochemistry and Molecular Biolo- gy-Universidade Federal do Parana ´ - Curitiba, Brazil 3 Instituto de Fı´sica, Unicamp, Campinas, Brazil 4 BNLS-Synchroton light source, Upton, USA Copyright ß 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim