ABSTRACT
By using transgenic methodologies, we have produced a number of mouse/human chimeric hemoglobins containing adult mouse and human embryonic globin chains. A detailed analysis of the oxygen binding properties of these proteins identifies the dominant role played by the specific beta-type globin chains in the control of the oxygen binding characteristics. Further analysis traces the origins of these effects to alterations in the properties of the T states of these proteins. The human zeta/mouse beta chimeric protein has been crystallized, and its structure has been determined by X-ray diffraction to a resolution of 2.1 A with R (R(free)) values of 21.6% (24.9%). Close examination of the structure indicates that the subunit interfaces contain contacts which, although different from those present in either the parent human or the parent mouse proteins, retain the overall stabilizing interactions seen in other R state hemoglobins.
Subject(s)
Globins/chemistry , Globins/physiology , Hemoglobins/chemistry , Hemoglobins/physiology , Oxygen/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/physiology , Adult , Animals , Crystallography, X-Ray , Globins/genetics , Globins/metabolism , Hemoglobins/genetics , Hemoglobins/metabolism , Humans , Mice , Mice, Transgenic , Models, Molecular , Protein Binding/genetics , Software , Structure-Activity RelationshipABSTRACT
Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from alpha-thalassemia. alpha-Thalassemia is a disease in which there is a deletion of one or more of the four alpha-chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma(4) homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O(2) affinity than either adult (alpha(2)beta(2)) or fetal (alpha(2)gamma(2)) Hbs, it has a lower affinity for O(2) than HbH (beta(4)). To better understand the association and ligand binding properties of the gamma(4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 A resolution (R = 21.1%, R(free) = 24.4%), and that of ferric azide (N(3)(-)) Hb Bart's was similarly determined at 1.86 A resolution (R = 18.4%, R(free) = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually long Fe-C bond of 2.25 A. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112 degrees, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.
Subject(s)
Hemoglobins, Abnormal/chemistry , Hemoglobins, Abnormal/metabolism , alpha-Thalassemia/metabolism , Azides/metabolism , Carbon Monoxide/metabolism , Crystallography, X-Ray , Heme/chemistry , Heme/metabolism , Humans , Ligands , Metals/metabolism , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits , Static Electricity , StereoisomerismABSTRACT
A variety of human haemoglobins (Hbs) are produced at different stages of human development, including three embryonic Hbs, foetal Hb and adult Hb. All are heterotetramers. During crystallization experiments on human embryonic Hb Portland (zeta(2)gamma(2)), it was discovered by crystallographic and biochemical analysis that the homotetramer Hb Bart's (gamma(4)) preferentially crystallizes from zeta(2)gamma(2) solutions below pH 5. This results from dissociation of Hb Portland into gamma(2) dimers and zeta monomers and has interesting implications for subunit interactions and tetramer stability in Hbs. It also makes possible a full crystallographic analysis of Hb Bart's, which is of considerable medical significance because of its presence in the red blood cells of millions of people worldwide who suffer from alpha-thalassaemia.
Subject(s)
Hemoglobins, Abnormal/chemistry , Chromatography, High Pressure Liquid , Crystallization , Crystallography, X-Ray , Humans , Hydrogen-Ion Concentration , Protein Conformation , SolutionsABSTRACT
The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents.
Subject(s)
Subtilisins/chemistry , Crystallography, X-Ray , Dimethylformamide/pharmacology , Dose-Response Relationship, Drug , Histidine/analysis , Magnetic Resonance Spectroscopy , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , ThermodynamicsABSTRACT
Histidine ammonium-lyase from P. putida was expressed in Escherichia coli, purified to homogeneity, and crystallized by the vapour-diffusion method using polyethylene glycol 3350 as the precipitant. The crystals, which diffract to at least 2.5 A resolution, exhibit the symmetry of space group P212121, with unit-cell parameters a = 89.7, b = 138.2 and c = 164.8 A. The asymmetric unit contains a tetramer, and the crystals have a Vm value of 2.41 A3 Da-1.
Subject(s)
Bacterial Proteins/chemistry , Histidine Ammonia-Lyase/chemistry , Pseudomonas putida/chemistry , Bacterial Proteins/isolation & purification , Crystallization , Crystallography, X-Ray , Histidine Ammonia-Lyase/isolation & purification , Protein Conformation , Pseudomonas putida/isolation & purificationABSTRACT
The Rhizobium meliloti DctD two-component receiver domain was expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using the hanging-drop vapor-diffusion geometry with ammonium phosphate as the precipitant. The crystals diffract to 2.3 A and exhibit the symmetry of space group I222 or I212121. The unit-cell dimensions are a = 59.0, b = 58.6 and c = 169.8 A. The asymmetric unit contains a dimer and the crystals have a Vm of 2.16 A3 Da-1.
