High-level expression and deuteration of sperm whale myoglobin. A study of its solvent structure by X-ray and neutron diffraction methods.
Neutron diffraction has become one of the best ways to study light atoms, such as hydrogens. Hydrogen however has a negative coherent scattering factor, and a large incoherent scattering factor, while deuterium has virtually no incoherent scattering, but a large positive coherent scattering factor. Beside causing high background due to its incoherent scattering, the negative coherent scattering of hydrogen tends to cancel out the positive contribution from other atoms in a neutron density map. Therefore a fully deuterated sample will yield better diffraction data with stronger density in the hydrogen position. On this basis, a sperm whale myoglobin gene modified to include part of the A c11 protein gene has been cloned into the T7 expression system. Milligram amounts of fully deuterated holo-myoglobin have been obtained and used for crystallization. The synthetic sperm whale myoglobin crystallized in P2(1) space group isomorphous with the native protein crystal. A complete X-ray diffraction dataset at 1.5A has been collected. This X-ray dataset, and a neutron data set collected previously on a protonated carbon-monoxymyoglobin crystal have been used for solvent structure studies. Both X-ray and neutron data have shown that there are ordered hydration layers around the protein surface. Solvent shell analysis on the neutron data further has shown that the first hydration layer behaves differently around polar and apolar regions of the protein surface. Finally, the structure of per-deuterated myoglobin has been refined using all reflections to a R factor of 17%.
Shu
F
F
Physics Department, State University of New York at Stony Brook 11794, USA.
Ramakrishnan
V
V
Schoenborn
B P
BP
D000818:Animals; D003460:Crystallization; D003461:Crystallography / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D003903:Deuterium; D004926:Escherichia coli; D007553:Isotope Labeling; D008958:Models, Molecular; D009211:Myoglobin / Q000096:biosynthesis / Q000737:chemistry*; D009502:Neutrons; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012542:Scattering, Radiation; D012997:Solvents; D014907:Whales
D009211:Myoglobin; D011993:Recombinant Fusion Proteins; D012997:Solvents; D003903:Deuterium