Structure determination is not always straightforward and membrane proteins present one of the many challenges that structural biology still faces. Membrane proteins are difficult to express, purify, and crystallise. Once crystals finally appear, they are often of poor quality and the modest number of known structures limits the use of molecular replacement techniques for solving the structure. Membrane proteins may be a challenge - but it can be done.
My main research interest is the P-type ATPases, a class of membrane embedded ion transporters, which are responsible for establishing and maintaining an asymmetric distribution of a number of cations across biological membranes. To perform this task they convert chemical energy, derived from ATP, into electrochemical gradients. Prominent members in eukaryotes of this primary transporter family are the Na+, K+- ATPases and H+-ATPases present in animal and plant cells, respectively. In addition, P-type ATPases are involved in gastric acid secretion (H+, K+- ATPase), the transport of heavy metals, and they have also been implicated in phospholipids metabolism as "flippases".
One of the members of the family is the sarcoplasmic reticulum (SR) calcium pump (Ca2+-ATPase) from skeletal muscle (SERCA 1a). It is a 112 kDa monomeric integral membrane protein, responsible for transporting calcium ions up the electrochemical gradient from the cytoplasm into the SR. By pumping calcium away from the cytoplasm, the Ca2+- ATPase terminates the muscle contraction induced by the preceding calcium release from the terminal cisternae via the dihydropyridine and ryanodine sensitive Ca2+ channels.
Studies of P-type ATPases relate to a very basic question in biology, namely how cation pumps work. Our crystal structures of Ca2+- ATPase have provided us with valuable insight into how P-type ATPases are able to convert the chemical energy, derived from hydrolysis of ATP, into a vectorial mechanism for cation transport.
My ongoing research focuses on the physiological regulation of SERCA activity, and on extending our understanding of the mechanism of ion pumps by looking at a number of other P-type ATPases involved in transport of other cations and heavy metals.
The structural biology pipeline from gene to structure is constantly being optimised and improved. Over the years many steps in the process have benefitted from developments in robotics, increased computing power and automated data analysis.
My beamline interests focus on development and implementation of techniques to extract information from challenging samples. To do this one should consider analysing samples and collecting data at room temperature, changing the crystal hydration state using humidity control devices, fluorescence or UV/Vis spectroscopy of samples to determine metals and chromophore content and their state. Implementing these techniques within the beamline is key to progressing with challenging targets.