124 research outputs found
Mutagenesis of the crystal contact of acidic fibroblast growth factor
Several mutations at Glu81 located on the crystal contact of human acidic fibroblast growth factor were studied in an effort to improve crystal growth. Mutation to Ser and Thr resulted in crystallization of a rather bulky form of the wild type, whereas mutation to Val prohibited crystallization. These results suggest that crystal growth may be controlled by designing a new interface by protein engineering
Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes : Insight into a Role of the Coiled-Coil Region
The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction
New data reduction protocol for Bragg reflections observed by TOF single-crystal neutron diffractometry for protein crystals with large unit cells
New data reduction protocol for Bragg reflections observed by TOF single-crystal neutron diffractometry for protein crystals with large unit cells
In protein crystallography, high backgrounds are caused by incoherent scattering from the hydrogen atoms of protein molecules and hydration water. In addition, the scattering intensity from large unit-cell crystals is very small, which makes it difficult to improve the signal-to-noise ratio. In the case of time-of-flight (TOF) single-crystal neutron diffractometry, the measured spectra cover four-dimensional space including X, Y, and TOF in addition to intensity. When estimating the integrated intensity, 3D background domains in the vicinity of peaks should be clearly classified. In conventional 1D or 2D background evaluation, the evaluation is applied for individual peaks assigned using peak searches; however, it is quite difficult to classify the 3D background domain in TOF protein single-crystal neutron diffraction experiments. We undertook the development of a data reduction protocol for measurements involving large biomacromolecules. At the initial stage of the reduction protocol, appropriate 3D background estimation and eliminations were applied over the entire range of X, Y, and TOF bins. The histograms were then searched for peaks and indexed, and the individually assigned peaks were finally integrated with an effective profile function in the TOF direction. Three-dimensional deconvolution procedures for overlapping peaks associated with large unit cells were implemented as necessary. This data reduction protocol may lead to the improvement of signal-to-noise ratios to enable TOF spectral analysis of large unit-cell protein crystals
Structural biology relating to electron transfer
Most electron transfer proteins have cofactor molecules, such as heme, flavin, and iron-sulfur clusters. Electron transfer reactions are regulated by the redox states of the cofactors, and hydrogen atoms are often involved in the conversion of redox states. Therefore, it is important to obtain precise structure information including hydrogen atoms for understanding the electron transfer reactions. We have determined structures of some electron transfer proteins, cytochrome b5 (b5; binding a heme cofactor) and NADH-cytochrome b5 reductase (b5R; binding a FAD cofactor) at the resolutions higher than 1.0 Å by X-ray crystallography. Structure information of some hydrogen atoms were clarified by these X-ray analyses. On the other hand, the rest of the hydrogen positions are still ambiguous.Neutron crystallography is a powerful technique to obtain accurate positions of hydrogen atoms in protein structures. Recently, we have performed high-resolution neutron and X-ray crystal structure analyses of b5R. b5R catalyzes the electron transfer from two electron carriers of NADH to one electron carrier of b5 and participates in fatty acid synthesis, cholesterol synthesis, and xenobiotic oxidation as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, b5R also participates in the reduction of methemoglobin. We succeeded in data collection of b5R (oxidized form) at high resolutions, 1.40 Å (at iBIX in J-PARC) and 1.45 Å (at BIODIFF in FRM-II), under cryogenic conditions. We have observed a hydrogen bonding network from FAD to His49, which is the only polar residue in a cluster of hydrophobic residues on the surface near FAD, so it seems to be responsible for electron transfer to b5. In addition, we have determined high-resolution X-ray crystal structures of the reduced form of b5R using wild-type and T66V mutant. The electron density map of the NAD cofactor clearly displays NAD+ and NADH states in wild-type and mutant, respectively. The neutron and X-ray structure analyses provide information about the hydrogen transfer pathway in b5R.3rd QST International Symposiu
Neutron crystallography for the elucidation of enzyme catalysis
Hydrogen atoms and hydration water molecules in proteins are indispensable for many biochemical processes, especially enzymatic catalysis. The locations of hydrogen atoms in proteins are usually predicted based on X-ray structures, but it is still very difficult to know the ionization states of the catalytic residues, the hydration structure of the protein, and the characteristics of hydrogen-bonding interactions. Neutron crystallography allows the direct observation of hydrogen atoms that play crucial roles in molecular recognition and the catalytic reactions of enzymes. In this review, we present the current status of neutron crystallography in structural biology and recent neutron structural analyses of three enzymes: ascorbate peroxidase, the main protease of severe acute respiratory syndrome coronavirus 2, and copper-containing nitrite reductase
Hydration and its hydrogen bonding state on a protein surface in the crystalline state as revealed by molecular dynamics simulation
Protein hydration is crucial for the stability and molecular recognition of a protein. Water molecules form a hydration water network on a protein surface via hydrogen bonds. This study examined the hydration structure and hydrogen bonding state of a protein, staphylococcal nuclease, at various hydration levels in its crystalline state by all-atom molecular dynamics (MD) simulation. Hydrophilic residues were more hydrated than hydrophobic residues. As the water content increases, both types of residues were uniformly more hydrated. The number of hydrogen bonds per single water asymptotically approaches 4, the same as bulk water. The distances and angles of hydrogen bonds in hydration water in the protein crystal were almost the same as those in the tetrahedral structure of bulk water regardless of the hydration level. The hydrogen bond structure of hydration water observed by MD simulations of the protein crystalline state was compared to the Hydrogen and Hydration Database for Biomolecule from experimental protein crystals
The Challenge of Visualizing the Bridging Hydride at the Active Site and Proton Network of [NiFe]‑Hydrogenase by Neutron Crystallography
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