Biomedical subjects
Publications and source records attributed to G Zanotti.
Crystals of hen eggwhite riboflavin-binding protein have been grown by equilibrium dialysis in solutions buffered with 0.05 M-Tris X HCl (pH 8.5) using ammonium sulphate as the precipitant. The crystals belong to the space group P3121 (or enantiomorph) with a = b = 112.5 A and c = 72.0 A, and diffract to a resolution of 2.8 A.
Crystals of three forms of human plasma apo-retinol-binding protein have been obtained using the procedure described for the holoprotein. The apoprotein was prepared by a novel method, which uses hydrophobic interaction and immobilized dye chromatography. The three forms were separated by fast protein liquid chromatography. All of the crystals are isomorphous and diffract to 2.5 A resolution. These crystals will be useful for studies of the mechanism of binding of retinol to its carrier using X-ray diffraction techniques.
The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.
Decompensated arthrosis secondary to genu varum is always a severely disabling condition. In English speaking countries there is an increasing trend towards prosthetic replacement surgery, but the present paper aims to show that valgising tibial osteotomy is still a valid form of treatment. The authors also discuss the various techniques that have been used and describe the advantages and results of their own preferred technique of "domed" osteotomy.
The frequency of operations on the knee, particularly on the antero-medial aspect, involving damage or section of sensory nerves, prompted the authors to carry out an accurate research on the innervation of this region. This revealed many variations of the normal anatomy which have been classified and illustrated in detail.
The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.
Crystals of human plasma retinol-binding protein have been obtained from 4.5 M-NaCl buffered at pH 6.8 with 20 mM-cacodylate. The crystals are trigonal with space group R3 and unit cell dimensions, referred to the hexagonal system, a = b = 104.2 A and c = 74.5 A. The crystals diffract to a resolution of 2.0 A.
In order to investigate the relative formation tendency of different tautomeric ring systems (cyclols, cyclodepsipeptides and corresponding N-acyl-diketopiperazines), two linear peptide precursors containing proline as C-terminal residue, have been synthesized and subjected to cyclizing conditions. Boc-Ser-Phe-Pro-ONp (I) gave three isomeric cyclic compounds: Boc-Ser-Phe-Pro- (IV), N-(Boc-Ser)-cyclo-(Phe-D-Pro) (III) and the corresponding aza-cyclol (II). Starting from Hyb-Phe-Pro-ONp (V) two epimeric N(3-hydroxybutyryl)diketopiperazines (VI) and (VII) and the corresponding 10-membered cyclodepsipeptide (VIII) could be isolated. Crystal and molecular structure of VIII is reported. Crystals of VIII are orthorhombic, P212121 with a = 9.684, b = 22.985, c = 7.841, z = 4. The two peptidic bonds are cis with omega values of 6.6 degrees and - 18.1 degrees, whereas the lactonic bond is of transoid type. The pyrrolidine ring has C2-C gamma-exo conformation.
Recent results from the crystallographic studies on glycogen phosphorylase b at 2 A resolution are reviewed with special reference to other themes of the meeting. The structural similarity of the fold of 150 residues in phosphorylase to the observed in lactate dehydrogenase is discussed and the binding sites for NADH in phosphorylase are described. The binding of the potent inhibitor glucose-1,2-cyclic phosphate to phosphorylase b in the crystal has been studied at 3 A resolution. The results are compared with those previously obtained for glucose-1-phosphate and discussed with reference to proposals for a mechanism of catalysis that involves the essential cofactor pyridoxal phosphate.
The inhibition constants (Ki) of DNA-dependent RNA polymerase B (or II) from calf thymus were measured for eight synthetically obtained bicyclic amanitin-like thioethers, two R-sulfoxides, and two S-sulfoxides. These Ki values were compared with those of alpha-amanitin, its 6'-O-methylether Ia (an R-sulfoxide), the S-sulfoxide, the sulfone, the S-deoxo derivative (Id) of Ia, and several previously described amatoxins. The necessity of a beta-methyl side chain in position 3 and a hydroxy group in proline-2 was confirmed. Additionally, the presence of an isoleucine side chain in position 6 and the absence of a side chain in position 5 was recognized as important for binding to the enzyme. In the three sulfoxide samples examined, the R-diastereomer was found to be a stronger inhibitor than the S-form. The contribution of every structural element to biological activity has been discussed.
Ile3-amaninamide (3-R) and its diastereomeric sulfoxide (3-S) are obtained by oxidation of the bicyclic thioether peptide 2 by hydrogen peroxide in acetic acid. 2 was prepared by an intramolecular Savige-Fontana reaction of the linear octapeptide tert.-butylester 4 whose N-terminal Boc-Hpi residue on treatment with TFA loses the Boc group and reacts under thioether formation with the released cysteine-SH. The concomitantly deprotected carboxyl terminus is coupled intramolecularly with the free amino group of the secocompound 5 using the MA or DCCI method, thus forming the homodetic peptide ring. Compounds 3-R and 3-S agree very well with analog samples in chiroptical behavior. Thioether 2 and sulfoxide 3-R exert 50% inhibition of RNA polymerase II (or B) from Drosophila melanogaster in 10(-6) M solution whereas Ki of 3-S is about five times higher.
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Observations of extended peptide chains, whose direction is perpendicular to the fiber axis (cross-beta-structures) have hitherto been confined to fibrous proteins and to some synthetic polydisperse polypeptides of rather low molecular weight. This structure has now been found in some monodisperse linear homo-oligopeptides with aliphatic hydrocarbon side chains. X-ray diffraction photographs of t-Boc-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe show characteristic reflections for this form. In addition, the good orientation of suitably prepared specimens has enabled a fairly complete determination of the unit cell of the heptapeptides to be made. t-Boc-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe molecules are packed in a monoclinic lattice with a = 4.80, b = 34.5, c = 5.82 A, beta = 65.6 degrees and a = 4.80, b = 29.5, c = 5.82 A, beta = 65.6 degrees, respectively. It has not been possible to establish whether the arrangement of the chains within the sheets is parallel or antiparallel.
L-3a-Hydroxy-1.2.3.3a.8.8a-hexahydropyrrolo [2,3-b-]-indole-2-carboxylic acid (Hpi), obtained from L-tryptophan by oxidation with peroxyacetic acid (Savige, 1975), after introduction of the Boc-residue at N-1, is coupled with various glycine peptides of S-trityl-L-cysteine to give the Hpi-peptides 6(a-f). By treatment with absolute trifluoroacetic acid these peptides are converted by an intramolecular thiolysis to the monocyclic thioethers 7(a-f). Two of them, 7e and 7f, can be subjected to a second cyclization by the mixed anhydride method thus yielding the bicyclic tryptathionine heptapeptide 8e and octapeptide 8f. In their structures the bicyclic molecules resemble the mushroom phallotoxins and amatoxins, respectively. They show CD spectra closely related to the naturally occurring bicyclic peptides thus indicating conformational similarities. The CD spectra of the other cyclic peptides synthesized are also presented and discussed.
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The conformation and calcium binding properties of the bicyclic nonapeptide BCP2, cyclo-(Glu(1)-Ala(2)-Pro(3)-Gly(4)-Lys(5)-Ala(6)-Pro(7)-Gly(8))-cyclo-(1gamma --> 5epsilon) Gly(9), have been investigated by means of NMR spectroscopy. Interproton distances, evaluated by nuclear Overhauser effect (NOE) contacts, and straight phi angles, from (3)J(NH-alphaCH), have been used to obtain a feasible model for the BCP2-Ca(2+) (BCP: bicyclic peptide) complex by means of restrained molecular dynamics (RMD). The NMR analysis of the free peptide, carried out in CD(3)CN, shows the presence in solution of at least four conformers in intermediate exchange rate. The addition of calcium ions caused the appearance of a new set of resonances, differing from those observed for the free BCP2. A comparison with published data about the conformational behavior of the closely analogous peptide BCP3, differing from BCP2 for two Leu residues instead of two Ala residues in positions 2 and 6, shows that this simple substitution dramatically increases the peptide flexibility. On the contrary, upon calcium ion addition, both BCP2 and BCP3 reach a strictly close conformation, as strongly testified by the almost identical (1)H-NMR spectra exhibited by both peptides. The RMD molecular model of the BCP2-Ca(2+) complex, here reported, is a quite symmetric structure, presenting a three-dimensional cavity ideal for the binding of spherical cations. Four carbonyls from the main ring (Ala(2), Gly(4), Ala(6) and Gly(8)) point toward it, offering, together with the two carbonyls of the peptide bridge (Gly(9) and gammaGlu(1)), putative coordinations to the cation.
The evaluation of peptide structures in solution is made feasible by the combined use of two-dimensional NMR in the laboratory (NOESY) and rotating frames (ROESY), and by the use of molecular dynamics calculations. The present paper describes how both the NMR method and molecular dynamics calculations were applied to very rigid synthetic bicycle peptides that are analogues of natural amatoxins. The NMR theory, which allows the estimate of interatomic distances between interacting nuclei, is briefly discussed. The experimental data were compared with those of known solid-state structures. Three amatoxin analogues have been examined. Of these, one is biologically active (S-deoxo gamma[R] OH-Ile3-amaninamide) and its structure in the solid state has recently been worked out. The second and third analogues (S-dexo-Ile3-Ala5-amaninamide and S-deoxo-D-Ile3-amaninamide, respectively) are inactive and their solid-state structures are unknown. The data presented confirm the authors previous hypothesis that lack of biological activity of S-deoxo-Ile3-Ala5-amaninamide is due to the masking of the tryptophan ring by the methyl group of L-Ala and not to massive conformational changes of the analogue.