ABSTRACT
The structural changes associated to increasing exposure of intense X-ray synchrotron radiation for a wide range of model proteins are summarized in this review for two distinct methods of crystallization. The two types of crystals are respectively grown by the Langmuir-Blodgett (LB) nanotemplate method and by the classical hanging drop method to quantify their distinct radiation resistance. Changes in parameters like B factor and reflection intensity versus absorbed dose, along with changes in electron density maps, were monitored for both types of crystals. The six model proteins were studied and compared using four different beamlines, namely, the ID-13, ID14-2, ID23-1, and ID29 of the ESRF in Grenoble, keeping in mind the fundamentals knowledge of radiation damage being acquired in the last decade. Consistently crystals grown by LB nanotemplatebased method proved to be significantly more radiation stable when compared to crystals grown by the classical hanging drop method. Synchrotron Radiation and Structural Proteomics Edited by Eugenia Pechkova and Christian Riekel Copyright © 2012 Pan Stanford Publishing Pte. Ltd. www.panstanford.com
9.1 INTRODUCTIONThe increase of interest to protein crystals was related to their importance for determining the macromolecule structures by X-ray crystallography. Other methods (like NMR, etc.) have serious limitations in terms of protein size and can be used with great difficulties for structure determination of the complexes, which are particularly important for the understanding of protein functions. With the recent rapid progress in macromolecular microcrystallography using synchrotron radiation (Cusack et al., 1998; Riekel, 2000), it is clear that X-ray crystallography will remain the most important structure determination method for the foreseeable future. Most macromolecular structures are nowadays determined using synchrotron radiation, profiting from the brilliance and tune ability, both in wavelength and size, of the X-ray beam. The development of structural proteomics is limited mostly by the problem of (i) initial crystal production and (ii) by the quality of diffraction data collected — in particular it is important to limit the influence of X-ray radiation damage. The biggest challenge in protein crystallography is that only a small fraction of proteins have been crystallized until now (Protein Structure Initiative, NIGMS-NIH, Bethesda, USA). Despite recent advances in the field of protein crystallization, crystal growth remains the slowest step and critical in determination of protein structure. The crystallization problem may be partially solved by the use of high throughput nanodrop robotic crystallization systems, which significantly increases the number of the crystallization trials and decreases the amount of protein required, thus allowing full automation of largescale crystallization experiments. However, still in many cases, protein crystallization remains the bottleneck to protein structure determination, and many scientifically and industrially important proteins have not been crystallized to date. Almost all approaches to protein crystallization are based on the classical crystallization methods (Rosenberger, 1996) (e.g., vapor diffusion, batch), varying crystallization conditions. However, these methods often give rare and non-reproducible results, first of all to the big and/or partly non-soluble proteins (i.e., membrane proteins) and require a long empirical search for the optimal crystallization conditions, since for every protein the specific crystallization conditions have to
be determined. For these reasons protein crystallization is often called art instead of science (Pechkova and Nicolini, 2002). This demands the exploration of novel crystallization techniques and a microscopic understanding of all steps involved in crystallization processes.
