Working in the Department of Biochemistry at the University of Pretoria, Prof Wolf-Dieter Schubert heads one of only a hand-full of structural biology groups in South Africa.
Prof Schubert holds BSc, Honours and MSc degrees in Chemistry from the University of Cape Town (UCT), specialising in Chemical Crystallography for the latter two degrees. For his PhD in Structural Biology he moved to the Free University of Berlin in Germany. “At the time there were no active structural biologists in South Africa, so I had to go overseas”, he explains. After his PhD, his journey led him to a post-doctoral stint in Japan before accepting another post-doctoral position in Braunschweig (Brunswick), Germany.
Prof Schubert‘s professional career began in Germany as Group Leader in Structural Biology at the Helmholtz Centre for Infection Research from 2005 to 2009. In November 2009 he moved to the University of the Western Cape as Professor of Structural Biology in the Department of Biotechnology. From there he joined the University of Pretoria In July 2013 to continue his research in the structural analysis of disease related proteins.
According to Prof Schubert, structural biology as a research field remains under-developed in South Africa. Universities in Europe or America often have entire departments of structural biology or multiple groups dispersed in, and collaborating closely with, different departments. “South Africa universities instead perceive structural biology as too expensive without recognising the long-term benefits. Fortunately, though, this is starting to change”, he says.
As a discipline, structural biology is an excellent tool for student teaching. “It really helps students to achieve a fundamental understanding of how biology works at the molecular and even the atomic level. It reveals the details of chemical reactions and how proteins bind other molecules such as metabolites, DNA, RNA or other proteins. A lot of what we know about biology has been influenced by structural biology without biologists realising it. Once a structure has been solved explaining a critical part of biology, people often forget how they came to understand the principle in the first place”, says Prof Schubert.
Techniques used for structural biology include X-Ray crystallography, nuclear magnetic resonance spectroscopy (NMR) and electron microscopy – with one or two experts for each within South Africa. Prof Schubert’s group mainly concentrates on X-ray crystallography. “We tend to combine structural analysis with biophysical characterisation such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) spectroscopy. These techniques tell us how specifically and how tightly molecules interact with each other”, he elaborates.
Prof Schubert and his group primarily use structural biology to answer questions about infectious diseases, with tuberculosis (TB) and listeriosis being two such diseases. Listeriosis is a food-borne disease. However, as it is not a notifiable disease in South Africa available data is quite limited. However, Listeria monocytogenes – the bacterium causing this infection – is a ‘model intracellular organism’ and has been well-characterised over time. “Its infection cycle is understood in some detail and our work thus fits into a large body of existing knowledge”, he says.
TB, by comparison, is not well understood at the molecular level. “Mycobacterium tuberculosis is a slow grower”, Prof Schubert explains. “This limits our options in trying to understand how TB infections work. We are currently concentrating on its metabolism. We aim to find critical enzymes that we can investigate and develop new inhibitors for. These may weaken the bacterium sufficiently to allow the human immune system to kill it off”. Collaborators on these TB-related projects include, ACGT partner, the CSIR.
Prof Schubert admits that crystallography is not a rapid technique. “You sometimes spend quite a bit of time overcoming hurdles on the way to adding another piece to the larger picture. In many ways structural biology is the opposite of the ‘‑omics’ revolution. In the latter, you collect large amounts of data about the ‘big picture’ and try to identify logical connections and trends in that data. We instead look at the individual molecule to assemble the larger picture in the end”. For an MSc or PhD thesis, a student will normally work on just one or perhaps two proteins.
The element of care and meticulous work helps to lay the foundation of a well-rounded training for his students, says Prof Schubert. “The overall training is universal because it covers a wide range of fields – such as molecular biology, computer science, chemistry and physics”.
Starting from the molecular biology angle, their work involves both the gene and the protein and identifies the part that is relevant to the project. “Very often we don’t work with the entire protein, but rather choose a critical domain. We clone the encoding DNA into expression vectors and transfer it into bacteria or another expression system”, he explains. The team then grows the microorganisms to generate tens to hundreds of milligrams of the protein. In biological terms this is a large amount, and is necessary because crystallisation requires that the protein sample is very pure. “Students are forced to work carefully in a very methodical, clean and sterile way, to eventually get down to minute details”, he says.
Following this process, the protein is crystallised. Once crystals have been optimised, a well-shaped crystal will be lifted out of the crystallisation drop and rapidly cooled to about 100 Kelvin or -273°C. This is necessary to prevent the high-intensity X-Rays required to produce a diffraction pattern from breaking the crystals too quickly. This is the part of the process that exposes the students to elements of physical science.
Using special computer programmes, each spot in the diffraction pattern is assigned a unique code and its intensity is digitally evaluated. After applying some special techniques, the distribution of the electrons in the crystal can then be calculated and displayed. Using this map a model of the protein can be built and refined to provide a precise position of each atom. “Proteins are quite big and normally each model will consist of several thousand atoms”, says Prof Schubert.
“Once we’ve located all atoms, we can then take a step back and ask: ‘What is this crystal structure telling us? Does this allow us to answer the initial question about the way the protein works; and how does this fit into the bigger picture?’”
Related to this bigger picture view is Prof Schubert’s opinion of the ACGT. “The Centre’s mission of initiating communication between different disciplines – in particular those requiring large infrastructure – is a very good thing”, he says. In his opinion, the ACGT plays a critical coordinating role. “Their work in organising multiple platforms to build expertise, pull efforts together and make people aware of funding opportunities and sources is critical”. “There is funding available internationally, but it’s not always clear to the researchers how to access that money, where the money is, and whether it is applicable to us or not”, he says.
For Prof Schubert, the ACGT’s role in coordinating the sharing of equipment and infrastructure is invaluable. “I think it’s also good that the Centre creates spaces where people can collectively decide what the most important pieces of infrastructure and equipment are – rather than individuals going off and buying their ‘favourite toys’. That type of coordination helps to get the biggest impact for the money that is being spent”, he concludes.