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A new X-ray technique, time-resolved wide-angle X-ray scattering (TR-WAXS) could defeat even high-field NMR spectroscopy in allowing researchers to monitor very fast, nanosecond-scale movements in the context of the overall three-dimensional protein structure. As a proof of principle, its developers have obtained new insights into the rapid structural changes that occur in the well-known oxygen-transport protein haemoglobin. Marco Cammarata, Friederike Ewald, and Michael Wulff of the European Synchrotron Radiation Facility (ESRF), in Grenoble, France and Jungkweon Choi and Hyotcherl Ihee of the Korea Advanced Institute of Science and Technology, in Daejeon, Republic of Korea, and colleagues Matteo Levantino and Antonio Cupane of the Department of Physical and Astronomical Sciences, at the University of Palermo, Italy, Friedrich Schotte and Philip Anfinrud of the Laboratory of Chemical Physics, at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, in Bethesda, Maryland, explain how viewing protein structure changes nanosecond resolution is possible in the current issue of Nature Methods. At their simplest, proteins are dynamic macromolecules. Functionality is intrinsic to their form, but their shifting shape is key to how they carry out their biological functions, whether they are acting as a biochemical catalyst, transporting other chemicals, or working as chemical sensors. Structural changes can range from the subtlest twitch of a single amino acid to the convulsive inversion of an active site. Structural changes can also occur slowly or so fast that until now they remained a blur to even the fastest visualisation techniques. Cammarata and colleagues point out that various methods for monitoring these protein changes, such as optical spectroscopy, are very good at detecting fast changes but do not yield much information about the overall three-dimensional protein structure. Other methods, such as nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, are very good at looking at the three-dimensional structures at different stages of the process, but have limited ability to detect very fast motions. TR-WAXS could be the best compromise for protein scientists. "Small-angle X-ray scattering probes the overall size and shape of the protein whereas wide-angle X-ray scattering (WAXS) gives more detailed information such as the fold of helices and sheets," they explain. A 160 microsecond barrier confronts these techniques because of the limitations of the detectors' frame rate. NMR suffers from being adequate only for smaller proteins and requiring complex radiolabelling to be truly effective. Moreover, its time resolution is of the order of milliseconds. In contrast, "TR-WAXS is complementary to time-resolved optical spectroscopy as it allows for tracking of tertiary and quaternary structural changes of a protein with global sensitivity; it is sensitive to changes in the position of all the atoms in the protein rather than to modifications around a given spectroscopic marker," the researchers explain. The team has used TR-WAXS to investigate one of the puzzling features of human haemoglobin (Hb). This tetrameric protein is comprised of two identical alpha-beta dimers that adopt at least two different quaternary structures in solution. The first is a relaxed (R) structure, which is stabilized usefully by dioxygen or lethally by carbon monoxide. The second structure is a tense (T) version that exists when no ligand is bound to the iron at the core of the protein's central haem group. "The transition among these two conformations plays a key role in the biological task of Hb i.e. efficient transport of oxygen from the lungs to the tissues," Cammarata explains. The switch between the R and the T forms involves not only conformational changes within the subunits (a tertiary transition) but also shifting of the positions of the subunits relative to each other, quaternary structure transition, in other words. The whole process is recognised as the epitome of biomolecular cooperation. The new TR-WAXS data presented in Nature Methods, help explain the switch between R and T forms. The researchers suggest that their approach, aside from working very well with the large test protein, haemoglobin, will also be amenable to a wide range of biologically relevant systems in solution. Indeed, the team has already carried out preliminary studies on sperm whale myoglobin (Mb) and the enzyme horse heart cytochrome c (Cyt-c). They add that the Mb data show just how sensitive TR-WAXS can be to local tertiary conformational changes, while the Cyt-c results reveal that the technique can also analyse the process of protein folding. Reference:
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