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M F Perutz

Publications and source records attributed to M F Perutz.

At least 73 records · Page 4Linked to original sources
PubMed1987

Iron distances in hemoglobin: comparison of x-ray crystallographic and extended x-ray absorption fine structure studies.

A comparison is presented of the structures obtained around the iron atom in deoxyhemoglobin (Hb). The data come from extended x-ray absorption fine structure (EXAFS) studies of the iron, which gave Fe-porphyrin nitrogen distances of 2.06 +/- 0.01 A, and from the most recent high-resolution x-ray crystallographic study, which gave exactly the same distance--2.06 +/- 0.02 A. The distance of Fe above the plane of the porphyrin nitrogens was 0.38 +/- 0.04 A from the crystallographic study; this value is not far from the upper limit of the distances 0.20 +/- (0.10)0.20 A calculated from the EXAFS experiment by triangulation. These distances above the nitrogen plane are shorter than those estimated in the earliest x-ray structures.

Hemoglobins↗
PubMed1987

X-ray crystallographic and functional studies of human haemoglobin mutants produced in Escherichia coli.

Human beta-globin was produced in Escherichia coli as a cleavable fusion protein using the expression vector pLcII [Nagai and Thøgersen, Nature 301, p. 810, 1984]. The fusion protein CIIFX beta-globin was purified under denaturing conditions to homogeneity and the authentic beta-globin was liberated by blood coagulation factor Xa. beta-Globin was then folded and reconstituted with haem and alpha-subunits to form fully functional alpha 2 beta 2-tetramers [Nagai et al., Proc. natn. Acad. Sci. USA 82, p. 7252, 1985]. This has enabled us to produce mutants with amino acid substitutions in the beta-subunit at will and in sufficient quantities to study their oxygen-binding properties and three-dimensional structures. We have crystallised three mutants, Hb Nympheas [Cys-93 beta----Ser], Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg] and Hb Sandra [Cys-93 beta----Ser, Asp-94 beta----Glu], and have solved their structures to high resolution by x-ray crystallography.

Amino Acid Sequence↗
PubMed1985

Comparison of histidine proton magnetic resonances of human carbonmonoxyhaemoglobin in different buffers.

We have recorded the C-2 proton resonances of the histidines of carbonmonoxyhaemoglobin A and of four abnormal human HbCOs in different buffers and at different concentrations of haemoglobin. Resonance H assigned by Perutz et al. (1985) to His HC3(146) beta, is present at both pH 7.30 and pH 6.90, but somewhat broadened when recorded in 5 to 10% HbCO A in 0.1 M-bis-Tris. The broadening disappears on tenfold dilution of the Hb with bis-Tris and the resonance then stands out sharply. Resonance H is absent at both Hb concentrations in HbCO Cowtown (His HC3(146) beta----Leu). HbCO Fort de France (His CD3(45) alpha----Arg) in 0.1 M-bis-Tris of pH 6.90 has a spectrum similar to that of HbCO A. In the same buffer a resonance marked L by Russu et al. (1982) is absent from the spectrum of Hb Abbruzzo (His H21(143) beta----Arg), whereas resonance H is present. Hb Barcelona contains an additional histidine in position FG1(94) beta; in 0.1 M-bis-Tris buffer of pH 6.90 its resonance is not resolved and resonance H is either shifted or broadened. The resonances of both histidines are resolved in phosphate buffer. At pH 6.90, spectra in 0.1 M-bis-Tris buffer are similar to those previously recorded in 0.2 M-HEPES. Addition of 0.1 M-KCl produces marked changes. Replacement of bis-Tris by 0.2 M-KCl + 0.2 M-phosphate gives rise to a different and much better resolved spectrum.

Buffers↗
PubMed1985

The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices.

Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 M-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146) beta. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94) beta, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 M-NaCl + 0.2 M-phosphate this change of ligation lowered the pKa of the histidine from 8.0 in Hb to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pKa in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect. We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)beta----Leu), HbCO Wood (His FG4(97)beta----Leu) and HbCO Malmø (His FG4(97)beta----Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)beta in fact belongs to His FG4(97)beta. Although in Hb the pKa of His HC3(146)beta is 8.05 +/- 0.05 independent of ionic strength, in HbCO its pKa drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pKa of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973). In HbCO A, His FG4(97)beta does not contribute to the Bohr effect, but in HbCO from which His HC3(146)beta has been cleaved (HbCO des-His), His FG4(97)beta is in equilibrium between two conformations with different pKa values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety. In HbCO A, His FG4(97)beta has a pKa of 7.8 compared to the pKa value of about 6.6 characteristic of free histidines at the surface of proteins. This high pKa is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of alpha-helices, which manifests itself even when they lie on the surface of the protein.

Carboxyhemoglobin↗
PubMed1985

Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli.

Human beta-globin was synthesized in Escherichia coli as a cleavable fusion protein, using the expression vector pLcIIFX beta-globin [Nagai, K. & Thøgersen, H. C. (1984) Nature (London) 309, 810-812]. The fusion protein cIIFX beta-globin was purified to homogeneity and cleaved at the junction by blood coagulation factor Xa; the authentic beta-globin was liberated. Beta-globin was folded in vitro and reconstituted with heme and alpha subunits to form alpha 2 beta 2 tetramers. The oxygen binding properties of reconstituted Hb are essentially the same as those of human native Hb. Two mutant Hbs (Hb Nymphéas [Cys-93 beta----Ser] and Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg]) were constructed by site-directed mutagenesis using synthetic oligonucleotides. Hb Nymphéas showed a slightly increased oxygen affinity and diminished cooperativity with normal 2,3-diphosphoglyceric acid and slightly reduced alkaline Bohr effects. Hb Daphne showed low cooperativity with high oxygen affinity. The alkaline Bohr effect was slightly reduced but the diphosphoglycerate effect was enhanced by 50% by the His-143 beta----Arg mutation. As arginine is fully charged at physiological pH and has a long flexible side chain, diphosphoglycerate binds more strongly to Hb Daphne.

Amino Acid Sequence↗
PubMed1984

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study.

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.

Adenosine Triphosphate↗
PubMed1984

The crystal structure of human deoxyhaemoglobin at 1.74 A resolution.

The structure of human deoxyhaemoglobin was refined at 1.74 A resolution using data collected on film at room temperature from a synchrotron X-ray source. The crystallographic R-factor is 16.0%. The estimated error in atomic positions is 0.1 A overall, 0.14 A for main-chain atoms of internal segments, and 0.05 A for the iron atoms. The effects of intermolecular contacts on the structure were investigated; such contacts cause only highly localized distortions, as judged from the degree of molecular asymmetry that they induce. The geometry of the iron-nitrogen complex closely resembles that of the deoxymyoglobin structure of Takano (1977) and of the 5-co-ordinated model compounds of Hoard (1975) and Jameson et al. (1980). The distance of the iron from the mean plane of N(porphyrin) is 0.40(5) A and 0.36(5) A, respectively, at the alpha and beta haems, in contrast to the corresponding distance of +0.12(8) A and -0.11(8) A in oxyhaemoglobin ( Shaanan , 1983); the Fe-N epsilon (F8) bond length is 2.12(4) A and the Fe-N(porphyrin) bond length is 2.06(2) A; the last is also in good agreement with extended X-ray fluorescence spectroscopy measurements on deoxyhaemoglobin ( Eisenberger et al., 1978; Perutz et al., 1982). The haems are domed toward the proximal side; the separation between the mean planes of N(porphyrin) and C(porphyrin) being 0.16(6) A and 0.10(6) A, respectively at the alpha and beta haems. At the alpha haems, the normals to the mean pyrrole planes are tilted uniformly toward the haem centre, by about three degrees relative to the haem normal, and there is a folding of about four degrees of the haem about an axis running between the methene carbons that are between the pyrrole rings bearing like-type side-chains. At the beta haems, there is no such folding, and only pyrroles II and IV (those eclipsed by His F8) are appreciably tilted, by about eight degrees. The independence of these parameters from restraints imposed on the model was verified by unrestrained refinement of the entire molecule starting from a structure with modified haem geometry.

Crystallography↗
PubMed1984

Structure of deoxyhemoglobin Cowtown [His HC3(146) beta----Leu]: origin of the alkaline Bohr effect and electrostatic interactions in hemoglobin.

Hemoglobin Cowtown [His HC3(146)-beta----Leu] exhibits high oxygen affinity and a halved alkaline Bohr effect. X-ray analysis shows the COOH-terminal leucine to be in equilibrium between two positions: one with the salt bridge between the terminal carboxyl and Lys C5(40)alpha intact and the leucyl side chain leaning against main chain atoms of helices F and FG and the other with the terminal salt bridge broken and the leucyl side chain touching Pro C2(37)alpha. Structural changes are confined to the immediate neighborhood of the COOH terminus, showing the halving of the alkaline Bohr effect to be due directly to the loss of the histidine, without significant contributions from changes in pK values of other ionizable groups due to structural changes elsewhere.

Amino Acid Sequence↗
PubMed1983

Physiological and x-ray studies of potential antisickling agents.

Several aromatic compounds have been found to inhibit the gelling of sickle cell hemoglobin. We have tried to correlate the antigelling activity of such compounds with the stereo-chemistry of their binding sites in the hemoglobin molecule. This approach led to the discovery that two known antilipoproteinemia drugs, clofibrate and gemfibrozil, have antigelling activity. X-ray analysis showed that three pairs of molecules of clofibric acid, the active metabolite of clofibrate, bound to the walls of the internal cavity of deoxyhemoglobin A; only one pair bound to a quite different site, between helices A, E, and H of the alpha chains of carbon monoxide hemoglobin A. Unlike other antigelling agents, clofibric acid and related compounds decrease rather than increase the oxygen affinity of hemoglobin.

Antisickling Agents↗
PubMed1983

Species adaptation in a protein molecule.

The allosteric properties of hemoglobins, especially their responses to ligands other than oxygen, vary widely in different classes of vertebrates. Knowing the stereochemistry of the cooperative effects in human hemoglobin, one can infer the stereochemical basis of these variations from the changes in amino acid sequence. The results indicate that the tertiary and quaternary structures of deoxy- and oxyhemoglobin have remained almost invariant during vertebrate evolution and that most of the amino acid replacements between species are functionally neutral. Adaptations leading to responses to new chemical stimuli have evolved by only a few (one to five) amino acid substitutions in key positions. Once such a response has become superfluous, it may be inactivated, not necessarily by a reversal of one of the original substitutions but by any other that happens to inhibit it.

Adaptation, Physiological↗
PubMed1982

Nature of the iron-oxygen bond and control of oxygen affinity of the haem by the structure of the globin in haemoglobin.

Spectroscopic and chemical evidence speak in favour of the iron-oxygen bond being polar. X-ray analysis shows that the oxygen molecule is inclined at an angle of about 115 degrees to the haem plane. Cooperative binding of oxygen by haemoglobin is attributable to an equilibrium between two alternative structures that differ in oxygen affinity by the equivalent of 3-3.5 kcal/mol. The author has proposed that in the low-affinity structure the globin opposes the movement of the iron atom from its pentacoordinated pyramidal geometry in the haem of deoxyhaemoglobin to its hexacoordinated planar geometry in the haem of oxyhaemoglobin, while in the high-affinity structure this restraint is absent. Recent evidence supporting this mechanism is described.

Animals↗