Deciphering enzyme mechanisms by neutron crystallography in combination with complementary methods

To understand the detailed function of biological systems, it is essential to use methods that can zoom in to an atomic and electronic level, and describe these systems in details. Here several different biochemical and biophysical methods need to be used. The main method used to obtain structural information about proteins and enzymes is X-ray diffraction, however, there is some limitations with this method. The X-rays used can damage the samples and lead to changes of the oxidation states, and in addition this method does not normally resolve the hydrogens in the structures. The hydrogens are essential to really understand the mechanism of many enzymes. Changing the radiation source from X-rays to neutrons, will overcome these problems, however, much larger protein samples (in this case crystals), and much longer data collection times are needed. This project will focus on building competence in using neutron diffraction to study proteins, and to combine this with X-ray diffraction and complementary spectroscopic methods as UV-vis and Raman spectroscopy. To do this both haem- and flavin protein system have been selected as model systems. The project intends through building competence in neutron diffraction to be able to answer some key biological questions about these protein model systems. For the haem system of myoglobin both sperm whale myoglobin and two myoglobin variants from gold fish have been cloned and expressed. We have crystal of myoglobin from horse heart, but are trying to discover a new crystallization condition that can give larger crystals. For the sperm whale and gold fish myoglobins, one variant has been purified and are in crystallization trials, while the second variant are poorly expressed and the third one seems to express an inactive variant. These issues are being resolved, but show the importance of working with different organisms. For the flavin systems we have crystallized, solved and published the X-ray structure of a novel flavin protein, and are now optimizing the crystallization conditions to be able to grow crystals suitable for neutron diffraction.

To be able to understand complex biological systems and how they function, it is essential that one can describe them on an atomic and electronic level. In biological processes proteins perform the chemical reactions in biological systems by acting as a catalyst and are involved in modification, build-up or break-down of different compounds needed by the cells. To enhance the understanding of these biological systems X-ray diffraction has to be combined with many different biochemical and biophysical methods. One limitation with X-ray diffraction is that hydrogens are normally not visible since the X-rays are scattered by the electrons, and therefore an essential information for deciphering reaction mechanism is missing. To be able to observe the hydrogens/protons, the X-rays can be exchanged with neutrons, which instead are scattered by the nuclei. In neutron protein crystallography, both hydrogen and deuterium are resolved since the hydrogen have a relative strong negative scattering length, while deuterium have positive scattering length. For metalloprotein and other redox cofactor proteins, the neutron diffraction have an additional advantage since it does not lead to radiation damage. Two main redox cofactor protein systems have been selected. One focusing on a haem protein system, and the other on a flavin protein system. We will through the neutron cryo-crystallography studies gain new insight into the peroxidase reaction in myoglobin and the electron transfer mechanism of flavodoxins. The ambition of the project will be to both developing competence in neutron protein crystallography, to develop the combination with X-ray protein crystallography, the combination with single-crystal spectroscopy, and deciphering the mechanism and function of some key redox cofactor enzyme systems.