New NMR strategy extends size limit and cuts cost

The research team at the Department of Biological Sciences is excited with a new breakthrough which has enabled them to study the structure of bigger proteins like the human adult haemoglobin. Analysing the high resolution structure of large proteins in solution like haemoglobins which have a molecular weight of about 65 kilodaltons (kDa) used to be impossible -- even with state-of-the-art NMR (nuclear magnetic resonance) technology.

However, with a new method that they have developed, the team is now able to determine structures of large proteins. Using this new strategy, the team is currently collaborating with the US to come out with the structure and dynamics for a protein which may one day make good artificial human blood.

NMR SPECIALIST: Associate Professor Yang Daiwen is rated as one of the world's best NMR spectroscopists. NMR spectroscopy offers high-resolution protein structure determination and is a relatively straightforward process for proteins smaller than 30 kDa. Characterising structures of larger proteins is painstaking and costly. However, the NUS team led by Associate Professor Yang Daiwen has solved this problem with a new NMR strategy. As most drug targets involve large proteins, solving the structures of these large proteins would enable pharmaceutical companies to design more effective drugs based on structures for the treatment of various diseases.

NMR technology involves a process where the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. Nuclei with the "spin" quality (which produces a small magnetic field) will enable NMR to pick up their signals. In principle, each hydrogen atom in a molecule will give rise to one specific signal. Associate Professor Yang explained that NMR spectra become increasingly complicated with the increase of amino acid residues found in bigger proteins. The conventional one-dimensional or two-dimensional NMR methods used for small organic compounds would not work.

Towards the end of the 1980s, scientists were able to obtain three- and four-dimensional NMR spectra with isotope enriched proteins -- but still, there were problems with large proteins. "This was unfortunate because in practice, many proteins are much bigger than for example, the 18-Da water molecule," said Associate Professor Yang.

The cost and time-intensive process for solving structures of large proteins in solution caused Associate Professor Yang to revisit the "conventional" NMR method for analysing the structure of small proteins. "I was thinking, if it worked for small proteins, it should somehow work for big proteins too. I just had to think of ways to get round the problems," said Associate Professor Yang.








His strategy did get away with deuteration (substitution of hydrogen to deuteron atom) as well as selective amino acid labelling (replacement of only some hydrogen atoms with deuterons in one amino acid) which are very costly and time-consuming but now commonly used for the study of proteins lager than 25 kDa.

Instead, his method is based on the signals received from all hydrogen atoms in the protein. Factoring in the spatial distances between hydrogen atoms derived from NMR signals, the "geometry" of the protein can thus be obtained. The challenge is, knowing which NMR signals correspond to which atoms, he said. This was overcome by using sensitive three- and four-dimensional NMR experiments to establish connectivity between two amino acid residues.

The team's discovery of the new method and findings were published in Nature Methods (November 2006). "This method when applied on small proteins can also cut the experimental time by up to 40 per cent," said Associate Professor Yang.