Analysis of Protein Crystallisation Parameters

Proteins are biochemical molecules that are essential for life processes. Their function is linked to their structure and so it follows an understanding of their structure will assist in an understanding of their function. The predominant method of solving protein structures is X-ray crystallography and for this a protein crystal is required. The process of obtaining a crystal is amongst the phases of the structure determination process with the highest rates of attrition. Analyses are performed throughout this thesis, which are intended to help improve output for this bottleneck. It has been possible to develop a method to determine pH using a spectrophotometer and acid-base indicator in an accurate, rapid and efficient manner. A method for predicting the pH of buffered solutions has also been developed and these predicted pH values are linked to the isoelectric point of a protein sequence. The isoelectric point is in turn used in classification, along with many other features, to determine a protein's propensity to crystallise. Finally, the most prevalent and successful chemical species in crystallisation are explored, compared and linked. These chemicals are used to design a new crystallisation screen

Predicting the effect of chemical factors on the pH of crystallisation trials

In macromolecular crystallisation, success is often dependent on the pH of the experiment. However, little is known about the pH of reagents used and it is generally assumed that the pH of the experiment will closely match that of any buffering chemical in the solution. We use a large data set of experimentally measured solution pH values to show that this assumption can be very wrong and generate a model which can be used to successfully predict the overall solution pH of a crystallisation experiment. Further, we investigate the time dependence of the pH of some polyethylene glycol polymers widely used in protein crystallisation under different storage conditions

A high-throughput colourimetric method for the determination of pH in crystallization screens

The crystallization of proteins is dependent on the careful control of numerous parameters, one of these being pH. The pH of crystallization is generally reported as that of the buffer; however, the true pH has been found to be as many as four pH units away. Measurement of pH with a meter is time-consuming and requires the reformatting of the crystallization solution. To overcome this, a high-throughput method for pH determination of buffered solutions has been developed with results comparable to those of a pH meter

Using isoelectric point to determine the pH for initial protein crystallisation trials

The identification of suitable conditions for crystallization is a rate-limiting step in protein structure determination. The pH of an experiment is an important parameter and has the potential to be used in data-mining studies to help reduce the number of crystallisation trials required. However, the pH is usually recorded as that of the buffer solution, which can be highly inaccurate. Results: Here we show that a better estimate of the true pH can be predicted by considering not only the buffer pH but also any other chemicals in the crystallisation solution. We use these more accurate pH values to investigate the disputed relationship between the pI of a protein and the pH at which it crystallises