Warner E. Love

Crystal structure of sickle-cell deoxyhemoglobin at 5 Å resolution

Crystals of sickle-cell deoxyhemoglobin were grown from solutions containing polyethylene glycol and citrate-phosphate buffer at a pH between 5 and 6. The crystals have the symmetry of the monoclinic space group P21, with a=63·28 A, b=184·19 A, c=52·84 A, and β=92·67°. The structure was determined by rotational and translational search procedures. Structure amplitudes and phases were calculated from the atomic co-ordinates of deoxy Hb§ A molecules appropriately positioned in the unit cell of the deoxy Hb S crystal. An Fo−Fo difference Fourier for the Hb S cristal was compouted at 5 A resolution. Portions of the Hb S molecules near the Val6β residues do not appear to be significantly different from the same portions of deoxy Hb A molecules crystallized in polyethylene glycol solutions at pH 7. In the Hb S crystal the molecular x axes enclose angle of less than 10° with the crystallographic a axis. The molecules are arranged in pairs of interlocking strands aligned with the a axis. The two strands in each pair are related approximately a 2-fold screw axis running between them longitudinally. Intermolecular contacts within each pair of strands involve Val6β and other residues that are believed to affect sickling interactions. Double strands, similar to those found in the Hb S crystal, can be incorporated into a fiber model that is consistent with available information on the structure of deoxy Hb S fibers in vivo.

Location of amino acid residues in human deoxy hemoglobin.

A table has been compiled of the spatial disposition of the amino acid residues in the human deoxy hemoglobin tetramer. The table also indicates regions of possible contact between residues in each subunit and possible contacts between subunits.

Refinement of a molecular model for lamprey hemoglobin from Petromyzon marinus.

A molecular model for the protein and ambient solvent of the complex of cyanide with methemoglobin V from the sea lamprey Petromyzon marinus yields an R-factor of 0.142 against X-ray diffraction data to 2.0 A resolution. The root-mean-square discrepancies from ideal bond length and angle are, respectively, 0.014 A and 1.5 degrees. Atoms that belong to planar groups deviate by 0.012 A from planes determined by a least-squares procedure. The average standard deviation for chiral volumes, peptide torsion angle and torsion angles of side-chains are 0.150 A3, 2.0 degrees and 19.4 degrees, respectively. The root-mean-square variation in the thermal parameters of bonded atoms of the polypeptide backbone is 1.21 A2; the variation in thermal parameters for side-chain atoms is 2.13 A2. The model includes multiple conformations for 11 side-chains of the 149 amino acid residues of the protein. We identify 231 locations as sites of water molecules in full or partial occupancy. The sum of occupancy factors for these sites is approximately 154, representing 28% of the 550 molecules of water within the crystallographic asymmetric unit. The environment of the heme in the cyanide complex of lamprey methemoglobin resembles the deoxy state of the mammalian tetramer. In particular, the bond between atom NE2 of the proximal histidine and the Fe lies 5.1 degrees from the normal of the heme plane. In deoxy- and carbonmonoxyhemoglobins, the deviations from the normal to the heme plane are 7 to 8 degrees and 1 degree, respectively. Furthermore, the inequality in the distance of atom CD2 of the proximal histidine from the pyrrole nitrogen of ring-C of the heme (distance = 3.29 A) and CE1 from the pyrrole nitrogen of ring-A (distance = 3.06 A) is characteristic of deoxyhemoglobin, not carbonmonoxyhemoglobin, where these distances are equal. Finally, a hydrogen bond exists between carbonyl 111 and the hydroxyl of tyrosine 149. The corresponding hydrogen link in the mammalian tetramer is central to the T to R state transition and is present in deoxyhemoglobin but absent in carbonmonoxyhemoglobin. We suggest that the low affinity of oxygen for lamprey hemoglobin may be a consequence of these T-state geometries.

Structure of deoxyhemoglobin a crystals grown from polyethylene glycol solutions

The structure of a new crystal form of deoxyhemoglobin A grown from polyethylene glycol solutions has been determined at 3·5 A resolution. The molecular orientations and positions were found by means of rotation and translation functions using the squared molecular transform of horse deoxyhemoglobin. Phases were calculated using atomic co-ordinates previously determined for deoxyhemoglobin A grown in another crystalline form. A difference Fourier synthesis showed minor structural differences near intermolecular contacts, the heme groups, and the β 1 carboxyl terminus. Some of these differences may be caused by the different crystalline environment; others may be due to errors in the analytical method. These apparent structural differences will be useful for interpreting results of a similar analysis of deoxyhemoglobin S crystals grown from the same solvent.

Crystal structure analysis of sea lamprey hemoglobin at 2 Å resolution

Abstract The crystal structure of the predominant hemoglobin component of blood from the sea lamprey, Petromyzon marinus, has been determined by X-ray diffraction analysis. Crystals for this analysis were grown from cyanide methemoglobin V as crystal type D2. These crystals are in space group P212121 and have unit cell dimensions of a = 44.57 A , b = 96.62 A and c = 31.34 A . Isomorphous heavyatom derivatives were prepared by soaking crystals in solutions of Hg(CN)2, K2Hg(CNS)4 and KAu(CN)2. Diffracted intensities to as far as 2 A spacings were measured on a diffractometer. Phases were found by means of the isomorphous replacements and anomalous scattering, with supplementary information provided by the tangent formula. An atomic model was fitted to the final electron density map in a Richards optical comparator. The lamprey hemoglobin molecule is generally similar in structure to other globins, but differs in many details. Each molecule is in contact with ten neighboring molecules in the crystal lattice. The nature of the binding of the heavy atoms to lamprey hemoglobin has been interpreted.

Structure of the Haemoglobin of the Marine Annelid Worm, Glycera dibranchiata, at 5.5 Å Resolution

THE wide, albeit sporadic, occurrence of haemoglobin among the phyla presents the opportunity to compare the structures of proteins with like functions but from dissimilar biological sources. Among the mammals myoglobin from the sperm whale1 and the seal2, and the subunits of horse3 and human4 haemoglobin, all have essentially the same tertiary structure. It has also been reported5 that haemoglobin from the fly, Chironomus thummi, has a similar structure.

The six hemoglobins of the sea lamprey (Petromyzon marinus)

Abstract The separation and purification of the six lamprey hemoglobins from Petromyzon marinus is reported. These six hemoglobins, which are single-chain, single heme, molecules (mol. wt. = 18,400), differ in their electrophoretic mobilities and isoelectric points. They show slight differences in their absorption spectra at different pH values, minor differences in solubility in strong salt solutions, and minor differences in stability at acid and alkaline pH. All these differences indicate that the primary structures of the six hemoglobins are different. Four of the six hemoglobins (1, 3, 4, 5) have been crystallized.

Structure of yellow fin tuna metmyoglobin at 6Å resolution

Abstract Using Patterson function searches we have shown that myoglobin from the yellow fin tuna ( Neothunnus macropterus ) has, at low resolution, a tertiary structure similar to that of sperm whale myoglobin. The position established for the heme iron by this procedure is in agreement with that independently derived from a Patterson synthesis based on anomalous dispersion.

Crystal forms of lamprey hemoglobin and crystalline transitions between ligand states

Abstract Hemoglobin from the sea lamprey, Petromyzon marinus , has been crystallized in nine basic forms having polymeric asymmetric units containing 1, 6, 8, 10, 12 or 16 monomers. This polymorphism and further variations within some of the forms are particularly dependent on temperature, salt of crystallization and ligand state of the heme iron. Additional variations in a form result when the ligand state is changed within the crystal. All the crystal types so far acquired, whether by growth or by intracrystalline transition, have been catalogued. Crystalline transitions between ligand states tend to fall into two classes. In the first case, both the crystal lattice and the intensity distribution change only a little. The transition product is essentially isomorphous with its parent. In the second case, the lattice parameters change by as much as 10% and the diffracted intensity is distributed quite differently. Changes of the ligand state in the monomeric form of lamprey hemoglobin are always accomplished isomorphously; only the polymeric forms can undergo non-isomorphous transitions. Changes of ligand state are thought to effect in some subtle way the modification of potential polymerization sites on the monomer without appreciable alteration of the protein conformation. In crystals with monomeric asymmetric units such changes would have little effect on the diffraction pattern. However, if the affected sites were actual points of contact between the subunits of polymeric crystals then, within the constraints imposed by lattice forces, a re-arrangement of the subunits with attendant changes in the diffraction pattern could ensue.

Crystals of Lumbricus erythrocruorin

Lumbricus terrestris erythrocruorin, a 3.9 X 10(6) Mr respiratory protein, has been crystallized in four different forms. Despite the high molecular symmetry apparent from images in electron micrographs, only one crystal form expresses any molecular symmetry as crystallographic symmetry. The lattice parameters provide upper limits on the molecular dimensions of 267 A X 308 A X 172 A (1 A = 0.1 nm), which agree well with dimensions obtained from electron micrographs of negatively stained molecules. We have collected diffraction data to 5.5 A from type III crystals and have begun a structural analysis.