A systematic approach to protein crystal growth and the investigation of intrinsic fluorescence for scoring protein crystals.

摘要

One of the most promising developments in treatment of molecular disease is structure based drug design. The success of this method depends on knowing the three-dimensional structure of the target macromolecules (usually proteins or enzymes) to a high degree of resolution. Typically, X-ray crystallography is the method used for structure determination. In order to perform X-ray analysis, a diffraction quality crystal must be grown. It is well known that protein crystal growth (PCG) is the bottleneck of the whole process of structure based drug design. The traditional method of growing protein crystals is screening and relies on trial and error. The Wilson Crystallization Slot (WCS) can be used to create a systematic strategy for growing protein crystals. In this work, regions of the WCS were explored to see if there was a correlation between the region of the slot and the number and size of the crystals grown. Three different regions of the slot were tested using lysozyme and ovalbumin crystals. Results for lysozyme show that a fewer number of larger crystals are obtained just inside the slot and a greater number of smaller crystals are obtained further into the slot.;This work also investigates intrinsic fluorescence for scoring protein crystals. Currently, there is no method for scoring protein crystals. A scoring assay would be a valuable tool to aid in the selection of crystals for X-ray diffraction analysis. Intrinsic fluorescence is a potential candidate for scoring protein crystals because it is a sensitive non-invasive technique. Crystals of lysozyme, glucose isomerase, thaumatin, thaumatin I, and ESA were analyzed using intrinsic fluorescence and X-ray analysis. Results show that there is a correlation between the fluorescence excitation maximum and the X-ray diffraction resolution limit for the protein crystals studied. A practical implication of using intrinsic fluorescence as a scoring tool for PCG is that crystals of common crystallizing agents do not fluoresce at wavelengths used in analysis of proteins. Intrinsic fluorescence could not only help researchers determine which protein crystals are suitable for X-ray diffraction analysis but also help discriminate between protein crystals and unwanted salt crystals.