research papers 330 doi:10.1107/S0909049509002763 J. Synchrotron Rad. (2009). 16, 330–335 Journal of Synchrotron Radiation ISSN 0909-0495 Received 14 April 2008 Accepted 22 January 2009 # 2009 International Union of Crystallography Printed in Singapore – all rights reserved MicroGISAXS of Langmuir–Blodgett protein films: effect of temperature on long-range order Eugenia Pechkova, a,b Shailesh Tripathi a,b and Claudio Nicolini a,b * a Nanoworld Institute-CIRSDNNOB, University of Genoa, Corso Europa 30, 16132 Genoa, Italy, and b Fondazione ElBA, Piazza SS Apostoli 66, 00187 Rome, Italy. E-mail: manuscript@nwi.unige.it The grazing-incidence small-angle X-ray scattering technique has been used here with a microfocus beamline (mGISAXS) to study the effect of temperature on the protein reorganization taking place in a Langmuir–Schaefer multilayered enzyme film. The study appears quite reproducible in the two enzymes being utilized, penicillin G acylase and urease. In-plane and out-of-plane cuts are used to account for the changes in the film thickness and distance between structures taking place by the process of heating up to 423 K and cooling to room- temperature. The out-of-plane cut suggests that the structures are getting closer and are becoming more organized owing to the heating affect. Merging of layers is likely to occur during the heating and cooling process, leading to a loss of correlation between the interfaces of the layers and to the establishment of long-range order. The dramatic increase in long-range order in the Langmuir– Blodgett multilayered enzyme films after heating and cooling, made here apparent by grazing-incidence small-angle X-ray scattering using a microbeam, could in the future open the way to avoiding the bottleneck of protein crystallization for protein structure determination. Keywords: penicillin G acylase; microGISAXS; urease; LB films. 1. Introduction Microbeam grazing-incidence small-angle X-ray scattering (mGISAX) is a powerful technique for investigating locally thin films and surfaces (Mu ¨ ller-Buschbaum et al., 2003; Roth et al., 2003; Pechkova et al., 2005; Nicolini & Pechkova, 2006a), giving access to length scales of up to several hundred nano- metres. The unique combination of a micrometre-sized beam and the reflection geometry allows us to gain, in principle, two orders of magnitude in spatial resolution compared with conventional GISAXS experiments, and thereby represents a very promising tool for studying biomolecular ordered systems, such as Langmuir–Blodgett (LB) thin protein films and crystals (Pechkova &Nicolini, 2003; Pechkova et al., 2005; Nicolini & Pechkova, 2006a). It was shown that the LB method, consisting of the formation and proper compression of protein monolayers at the air–water interface (Nicolini et al., 1993), appears capable of depositing the resulting protein monolayers onto solid substrates and to preserve protein structure and function, providing new useful properties such as protein temporal stability, film anisotropy and protein heat stability (Nicolini, 1997; Nicolini & Pechkova, 2006b). The secondary structure of all studied proteins in LB films is heat- proofed up to 473 K (for a review, see Nicolini, 1997; Nicolini & Pechkova, 2006b). A coherent explanation for the origin and the molecular mechanisms of the protein heat proof in LB films can be drawn by the comparative atomic structure characterization of thermophilic versus mesophilic proteins by X-ray crystallographic diffraction and nanogravimetric analysis in protein solution, thin films and crystals, recently pointing to the role of inner bound water in determining protein thermostability (Pechkova, Sivozhelezov & Nicolini, 2007, and references within). Moreover, it was shown for LB films of photosynthetic reaction centres that special thermal treatment can improve the film ordering (Facci et al., 1994). In order to further understand this phenomenon, it is necessary to study the changes taking place in the protein orientation and long- range order in thin films after heating. Though non-contact ‘tapping’ mode atomic force microscopy can be used as a topography-sensitive method to provide the surface image of a LB protein sample (Sartore et al. , 2000), scattering analysis can give better in-depth insight. Since the original synchrotron study on a heat-proof LB monolayer (Erokhin et al., 1998), much progress in X-ray scattering techniques has been made, namely at the ID13 beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble (Riekel, 2000) where the mGISAX set-up has been developed (Mu ¨ ller-Buschbaum et al., 2003; Roth et al., 2003). Now it is possible to investigate laterally inhomogeneous surfaces and interfaces with a two order-of-magnitude increase in spatial resolution compared with standard reflection set-ups and a