Development and application of novel crystallisation methodology to medically relevant proteins

Abstract

X-ray crystallography is the most widely used technique for determining 3-D protein structures. Information gained from crystallography is vital to the success of rational drug design and other biotechnological applications. Crystallography relies on the production of high quality protein crystals but obtaining such crystals is a major obstacle to progress. This project tackles this problem by (i) designing, developing and validating new and improved crystallisation methodologies for obtaining high quality crystals; and (ii) applying the new techniques in addition to existing methods, to crystallise medically important proteins in order to facilitate their 3-Dimensional structure determination.

Presented here is the design and validation of an improved method to slow down protein crystallisation in order to enable the growth of fewer, larger single crystals of higher quality. The method called the oil-on-drop method is a variation of the Chayen method which places an oil barrier over precipitant reservoirs in hanging drop trials. The variation consists of dispensing the oil directly onto the protein drops. This method was successfully tested and validated using five proteins, three of which are of medical relevance including a methyltransferase and an antibody-peptide complex.

A comparison of the oil-on-drop method with existing standard and non-standard techniques for optimising crystal quality such as the application of nucleants and the Chayen method showed the oil-on-drop technique to be superior in many ways. Most importantly, it reproducibly yielded crystals diffracting to higher resolution of three of the proteins tested namely the methyltransferase, the complex and trypsin. In the case of the Roab13 antibody-peptide complex the crystals obtained were of the highest resolution than ever attained previously. In addition, the oil-on-drop method overcomes problems encountered by other methods. It can be used with a wider variety of precipitating agents and can be performed using robots in a high throughput mode.