Molecular packing in a second monoclinic crystal of deoxygenated sickle hemoglobin

A close correspondence has been demonstrated between double filaments of deoxygenated hemoglobin S molecules as found in monoclinic crystals, forms I and II, and in sickle fibers. We have carried out a low resolution study of monoclinic form II by X-ray diffraction analysis. Its structure differs from that of form I solely by a shift along the a-axis of the molecular centers of the asymmetric unit, which forms the double filament. The magnitude of the translation was determined from a minimum residual calculation. The x co-ordinates of the symmetry related molecular centers of antipolar double filaments are approximately the same. This means that the double filaments are nearly in register. A minor component associated with form II crystals proved to be form I. The possible existence of additional forms is discussed.The significance of the molecular arrangement in form II is related to its presence in sickle fibers. We have determined the contacts between antipolar double filaments in this form as well as a number in form I not tabulated previously. These new contacts represent additional stabilizing interactions that might provide targets for the design of stereospecific antisickling agents.     

Molecular topology in crystals and fibers of hemoglobin S

Several lines of evidence indicate a close correspondence between the linear double filaments in the crystal form of hemoglobin S grown from solutions containing polyethylene glycol and the seven pairs of helical filaments that occur in the 14-filament fibers of hemoglobin S. An analysis of the adjustments to the intermolecular contacts required to convert the double filaments from crystals to fibers is presented here. In addition, postulated contacts between the helical double filaments, which are distinct from any of the contacts of the crystals, are specified for the first time. The movements from crystals to fibers are described in terms of three rotation angles: α, the inclination of the filaments with respect to the fiber axis; δ, the tilt of successive molecules along the filaments; and ω, the rotation of successive molecules along the filaments. On the basis of the fiber structure determined by three-dimensional reconstruction of electron micrographs and the assignment of filament pairs from data on incomplete fibers, the various angles have been evaluated. For the filaments at various radii in the fibers, a varies from 3 ° to 12 °, δ varies from 1 ° to 4 ° and ω is constant at 9 °. The effects of the rotations on the contacts between molecules of hemoglobin S at various positions in the fibers are characterized using surface maps based on polar coordinates. For each residue on the surface of hemoglobin the centroid position of its side-chain is located by a longitude, a latitude and an altitude. Locations on the maps are assigned for the contacts within the helical double filaments, as well as 11 classes of new contacts describing the potential interaction sites between double filaments. The resulting maps (1) deduce roles for the various α mutants of hemoglobin known to influence fiber formation that have been identified by the Benesches; (2) distinguish effects for the α chain mutants on the same (cis) or opposite (trans) α₁β₁ dimer as the β6 Val in asymmetric tetramers; (3) propose new sites where effects of mutations on fiber formation may be found; and (4) suggest why some mutants may inhibit, while others enhance, fiber formation. Concerning the last point, the possibility of certain mutants “correcting” the effects of other mutants is proposed as a test of contact assignments.     

Structural transformations in protein crystals caused by controlled dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.     

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.     

On the assembly of sickle hemoglobin fascicles

Deoxyhemoglobin S fibers associate into bundles, or fascicles, that subsequently crystallize by a process of alignment and fusion. We have used electron microscopy to study the formation of fascicles and the changes in fiber packing that occur during the conversion of fascicles to crystals. The first event in crystallization involves fibers forming fascicles that are initially small and poorly ordered but, with time, become progressively larger and more highly ordered. After six to eight hours, the fibers in a fascicle form a crystalline lattice. The three-dimensional unit cell parameters of this lattice are a = 1300 A, b = 365 A, and c = 210 A (the a axis is parallel to the fiber axis). Fibers have an elliptical cross-section whose major and minor axes are 250 A and 185 A, respectively. When projected on to the unit cell vectors, these dimensions are 210 A and 155 A, so the unit cell dimension of 365 A implies that there are two fibers per unit cell. Theoretically, fibers could pair so that each member of the unit cell is oriented in the same direction (parallel) or opposite directions (antiparallel). Fourier transforms of electron micrographs (or models) cannot distinguish between these alternatives, since the two arrangements produce very similar intensity distributions. The orientation of the fibers was determined from cross-sections of the fascicles in which the fibers are seen end-on. In this view the images of the fibers are rotationally blurred because the fibers twist 30 degrees to 40 degrees about their helical axis through the 300 A to 400 A thick section. We have been able to remove the rotational blur from each of the fibers in the unit cell using the procedures described by Carragher et al. The deblurred images of the two fibers in the unit cell are related by mirror symmetry. This relationship means that the fibers are antiparallel. These observations suggest that crystallization of fibers in fascicles is mediated by assembly of the fibers into antiparallel pairs that contain equal numbers of double strands running in each direction.     

Preliminary X-ray crystallographic study of methanol dehydrogenase from Methylophilus methylotrophus

Single crystals of methanol dehydrogenase from Methylophilus methylotrophus have been prepared by the macroseeding method. The crystals belong to the monoclinic space group C2, and have unit cell parameters a = 125.62 A, b = 63.83 A, c = 83.99 A, and beta = 93.24 degrees. There is one 62,000 Mr monomer in the asymmetric unit. The crystals diffract to beyond 2.0 A resolution.     

Crystal structures of the peanut lectin-lactose complex at acidic pH: retention of unusual quaternary structure, empty and carbohydrate bound combining sites, molecular mimicry and crystal packing directed by interactions at the combining site

The crystal structures of a monoclinic and a triclinic form of the peanut lectin-lactose complex, grown at pH 4.6, have been determined. They contain two and one crystallographically independent tetramers, respectively. The unusual "open" quaternary structure of the lectin, observed in the orthorhombic complex grown in neutral pH, is retained at the acidic pH. The sugar molecule is bound to three of the eight subunits in the monoclinic crystals, whereas the combining sites in four are empty. The lectin-sugar interactions are almost the same at neutral and acidic pH. A comparison of the sugar-bound and free subunits indicates that the geometry of the combining site is relatively unaffected by ligand binding. The combining site of the eighth subunit in the monoclinic crystals is bound to a peptide stretch in a loop from a neighboring molecule. The same interaction exists in two subunits of the triclinic crystals, whereas density corresponding to sugar exists in the combining sites of the other two subunits. Solution studies show that oligopeptides with sequences corresponding to that in the loop bind to the lectin at acidic pH, but only with reduced affinity at neutral pH. The reverse is the case with the binding of lactose to the lectin. A comparison of the neutral and acidic pH crystal structures indicates that the molecular packing in the latter is directed to a substantial extent by the increased affinity of the peptide loop to the combining site at acidic pH.     

Crystallization and preliminary x-ray crystallographic analysis of galectin LEC-1 from Caenorhabditis elegans

Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 A, alpha = 108.73, beta= 91.39, and gamma = 98.45 degrees and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 A, and beta= 113.30 degrees and one protein molecule per asymmetric unit.     

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

On the DNA binding protein II from Bacillus stearothermophilus. I. Purification, studies in solution, and crystallization

DNA binding protein II from Bacillus stearothermophilus has been purified as a single species from the nonribosomal cell fraction by a combination of gel filtration and ion exchange chromatography. The protein occurs in solution as a tetramer and is able to bind to 30 S, 50 S, and 70 S ribosomal particles. Circular dichroism studies show that the protein has approximately 45% alpha-helix. The secondary structure of the Bacillus protein is considerably more resistant to the effects of increasing temperature and urea concentration than the homologous protein (NS1 and NS2) from Escherichia coli. Proton magnetic resonance experiments show that the protein has a well folded, compact tertiary structure. The DNA binding protein has been crystallized from several precipitants as monoclinic needles and triclinic plates. The monoclinic form diffracts to at least 3.5 A and oscillation data from the native crystals have been collected. The protein is able to bind to both single- and double-stranded oligodeoxyribonucleotides. Upon binding, several changes occur in the protein NMR spectrum which may be used for further analysis of the mechanism of interaction.     

Characterization of crystals of an intact monoclonal antibody for canine lymphoma

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.     

The binding of calcium to glycerinated muscle fibers in rigor. The effect of filament overlap.

The binding of Ca2+ to glycerinated rabbit psoas fibers of varying sarcomere length was measured with a double isotope technique and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid buffers. Experiments were carried out under rigor conditions with fiber bundles pre-set at different lengths prior to extraction with detergent and glycerol. These experiments were designed to test whether rigor complex formation, determined by the degree of filament overlap, enhances Ca2+-receptor affinity in the intact filament lattice, as it does in reconstituted actomyosin systems. The Ca2+-receptor affinity, as indicated by the free Ca2+ concentration at half-saturation and by the slopes of Scatchard plots, was found to be relatively unaffected by variations in filament overlap. However, the maximum bound Ca2+ was significantly reduced in stretched fibers. With maximum filament overlap the bound Ca2+ was equivalent to 4 mol per mol troponin. When stretched to zero overlap the fibers bound a maximum of 3 mol Ca2+ per mol troponin. When fibers with maximum overlap were incubated in the presence of 5 mM MgATP there was a reduction in the number of Ca2+-binding sites equivalent to that caused by stretching the fibers. These findings, taken together with other data in the literature, suggest that in the intact filament lattice at least one of the Ca2+-binding sites is present only when cross-bridge attachments are formed.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

Crystallization of deoxyhemoglobin S by fiber alignment and fusion.

Fibers of deoxyhemoglobin S obtained directly from lysed sickled red blood cells have been compared with fibers from chromatographically pure deoxyhemoglobin S solutions of known chemical composition. Electron micrographs of negatively stained specimens reveal that the molecular packing within the fibers remains largely invariant with changes in pH, ionic strength, Mg²⁺ concentration, 2,3-diphosphoglycerate concentration, temperature or the method of deoxygenation.When solutions of chromatographically pure deoxyhemoglobin S are stirred, the fibers align into well defined fascicles. After several hours of stirring, long needles and twisted ribbons develop and in a relatively short time replace the fascicles in solution. With continued stirring all forms are replaced by small crystals. By use of electron microscopy and low-angle X-ray diffraction we have found these crystals to have cell parameters indistinguishable from those of crystals grown in polyethylene glycol and citrate/phosphate buffer at pH 5 to 6 (Wishner et al., 1975a).Our evidence indicates that crystal formation in stirred solutions of deoxyhemoglobin S is the result of a progressive alignment and fusion of the fibers, and that the molecular arrangement within the fibers is closely related to that within the crystal. The remarkable pH invariance of the molecular packing within the fiber and crystal structures is consistent with the dominance of hydrophobic bonding between molecules. The β6-valine contact observed by Wishner et al. (1975b) is apparently the pathological contact responsible for the polymerization of deoxyhemoglobin S in vivo. On the basis of our observations and knowledge of the crystal structure we propose that the deoxyhemoglobin S fiber consists of eight molecular double strands, four of which run in each direction along the length of the fiber.     

Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

Crystallization and characterization of the recombinant human Clara cell 10-kDa protein

Crystals of recombinant human Clara cell 10-kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X-ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 Å, b = 83.0 Å, c = 58.3 Å, and β = 99.7°. The monoclinic crystals diffract to beyond 2.5 Å. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 Å, b = 46.3 Å, c = 51.3 Å, α = 117.7°, β = 102.3°, and γ = 71.4°. These crystals diffract to beyond 3.2 Å. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.     

Crystallization and preliminary data for the ferric form of Lucina pectinata hemoglobin I.

Cytoplasmic monomeric hemoglobin I from the bacteria-harboring gill of the bivalve mollusc Lucina pectinata has been crystallized in a form suitable for atomic resolution X-ray structural investigations. The crystals have been grown at pH 4.8, in 0.05 M-acetate buffer, using 2.6 M-ammonium sulfate as precipitating agent. The crystals belong to the monoclinic space group P2(1), with unit cell constants a = 50.0 A, b = 38.6 A, c = 42.1 A, beta = 107.1 degrees, and contain one molecule (14,000 Mr) in the asymmetric unit. By means of single crystal microspectrophotometry it has been shown that the crystals contain the ferric form of L. pectinata "sulfide reactive" hemoglobin I. On the other hand, by careful control of the buffering medium composition, it has been possible to obtain stable crystals of the deoxy, oxy and sulfide forms of the protein.