Biomedical subjects
Publications and source records attributed to L Sawyer.
Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 2.3 A on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P2(1)2(1)2 with unit cell dimensions a = 48.01 A, b = 114.29 A, c = 42.87 A. There is one subunit in the asymmetric unit.
The alcohol dehydrogenase (ADHase) enzyme catalyses the oxidation of alcohols to aldehydes or ketones using NAD+ as a cofactor. Functional ADHase from Drosophila lebanonensis is a dimer, with a monomeric molecular weight of 27,000 and with 254 residues in each polypeptide chain. Crystals of the protein have been grown with and without NAD+. Two crystal forms have been observed. Most crystals are plate-like, 0.05 mm in their shortest dimension and up to 0.4 mm in their longest dimension. These crystals are generally too small to diffract efficiently using conventional X-ray sources, so preliminary studies were carried out using the Synchrotron Radiation Source at the SERC Daresbury Laboratory. Twinning was a severe problem with this crystal form. The second form is grown in the absence of NAD+ but with DL-dithiothreitol present. These crystals grow more evenly and diffract to better than 2 A resolution. They are monoclinic, with cell dimensions, a = 81.24(6) A, b = 55.75(4) A, c = 109.60(7) A and beta = 94.26(9) degrees, space group P2(1). There are two dimers in the asymmetric unit, but at low resolution a rotated cell with one dimer per asymmetric unit can be obtained.
The transglutaminase-catalysed incorporation of primary amines (putrescine and monodansylcadaverine) into bovine beta-lactoglobulin has been studied. In the presence of 1 mM-dithiothreitol between 1 and 2 mol of amine can be incorporated per mol of beta-lactoglobulin subunit. There is very little incorporation of amines in the absence of reducing agent. By isolating and sequencing the modified peptides, the sites of modification have been identified as Gln-159 (preferred) and Gln-155. C.d. has been used to study the structure of beta-lactoglobulin over a range of pH values and in the presence or absence of dithiothreitol. The results are discussed in terms of the X-ray-crystallographically determined structure of beta-lactoglobulin.
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The transglutaminase-catalysed incorporation of putrescine and monodansylcadaverine into yeast phosphoglycerate kinase has been studied. There is little incorporation of the amines into wild-type enzyme, but nearly stoichiometric incorporation into the histidine-388----glutamine mutant enzyme. C.d. studies show that the overall structure of the mutant enzyme is very similar to that of the wild-type enzyme. Incorporation of the amines into the mutant enzyme causes no significant change in its activity. Glutamine-388 was shown, by isolation and sequencing of the modified peptide, to be the site of incorporation of monodansylcadaverine into the mutant enzyme. The specificity of the transglutaminase reaction is discussed in the light of available data.
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Circular dichroism (CD) measurements on the coat protein of narcissus mosaic virus particles show that the dominant secondary structure is the alpha-helix, with a 45 (+/- 2)% content. The beta-sheet content is much lower at 5 (+/- 3)%. Both values are essentially the same as those found in potato virus X coat protein. The CD results are used to assess the results of secondary structure prediction methods.
7-(3-[1-(4-Phenylthiosemicarbazono)ethyl]-bicyclo[2.2.1]hept-2- yl)-5-heptenoic acid, C23H31N3-O2S, Mr = 413.7, monoclinic, P2(1)/a, a = 12.9650 (7), b = 11.2081 (6), c = 16.8941 (12) A, beta = 110.452 (5) degrees, V = 2300.2 A3, Z = 4, Dx = 1.194 g cm-3, Mo K alpha, lambda = 0.71073 A, mu = 1.55 cm-1, F(000) = 888, T = 298 K. Final R = 0.0472 with 2778 independent data. EP 092 is a thromboxane receptor antagonist akin to many other analogues of the thromboxane A2 [Wilson & Jones (1985). Adv. Prostaglandin Thromboxane Leukotriene Res. 14, 393-425].
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The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turns for fructose-bisphosphate aldolases from human and rat muscle and liver, from Trypanosoma brucei and from Drosophila melanogaster. All the aldolase sequences gave essentially the same pattern of secondary-structure predictions despite having sequences up to 50% different. One exception to this pattern was an additional strongly predicted helix in the rat liver and Drosophila enzymes. Regions of relatively high sequence variation generally were predicted as reverse turns, and probably occur as surface loops. Most of the positions corresponding to exon boundaries are located between regions predicted to have secondary-structural elements consistent with a compact structure. The predominantly alternating alpha/beta structure predicted is consistent with the alpha/beta-barrel structure indicated by preliminary high-resolution X-ray diffraction studies on rabbit muscle aldolase [Sygusch, Beaudry & Allaire (1986) Biophys. J. 49, 287a].
In free solution, the caseins behave as non-compact and largely flexible molecules with a high proportion of residues accessible to solvent. Historically, they have been described as random coil-type proteins with only a nutritional function. Nevertheless, secondary structure prediction algorithms indicate that many parts of the (unphosphorylated, unglycosylated) polypeptide chains can form regular structures. In particular, a recurrent motif of the Ca2+-sensitive caseins in man, rat, mouse, guinea pig and ruminant species is an alpha-helix--loop--alpha-helix conformation in which the loop region typically contains a cluster of sites of phosphorylation. The biological function of the caseins is considered and it is suggested that the potential or actual conformations of the group of Ca2+-sensitive caseins are suited to the function of modulating the precipitation of calcium phosphate from solution. Either they can act as sites for nucleation or they can bind rapidly to calcium phosphate nuclei as they form spontaneously from supersaturated solution.
The structure of the trigonal crystal form of bovine beta-lactoglobulin has been determined by X-ray diffraction methods. An electron density map, calculated with phases obtained by the multiple isomorphous replacement method, served as a starting point for alternate cycles of model building and restrained least-squares refinement. The model of the molecule fitted to the initial Fourier map was the one built for the orthorhombic crystal form of beta-lactoglobulin, solved at 2.8 A resolution (1 A = 0.1 nm). The final R factor for 1456 atoms (1276 non-hydrogen protein atoms and 180 solvent atoms) is 0.22, including 5245 reflections from 6.0 to 2.5 A. The molecule shows significant differences in the two crystal forms mentioned, mainly due to different packing. In the trigonal form, the species crystallized does not appear to be dimeric, but a linear polymer with tight intermolecular contacts. A difference electron density map between the complex of beta-lactoglobulin with retinol and the native protein shows no significant peaks in the cavity which, in the similar retinol-binding protein, binds the chromophore. Instead, differences are found at a surface pocket, which is limited almost completely by hydrophobic residues.
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The results of several secondary-structure prediction programs were combined to produce an estimate of the regions of alpha-helix, beta-sheet and reverse turn for both chicken skeletal-muscle and yeast enolase sequences. The predicted secondary-structure content of the chicken enzyme is 27% alpha-helix and less than 10% beta-sheet, whereas in the yeast enolase a similar helix content but virtually no sheet are predicted. These results are in fair agreement with published experimental estimates of the amount of secondary structure in the yeast enzyme. The enzyme appears to be formed from three domains.
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The preparation, crystallization and low-resolution structure determination of beta-lactamase (EC 3.5.2.6, 'penicillinase') from Staphylococcus aureus is described. The enzyme crystallizes in space group I222 with 1 molecule per asymmetric unit and cell dimensions a = 5.45(1), b = 9.39(1) and c = 13.87(2) nm. The structure was determined at 0.5 nm resolution by using phases calculated from (NH4)2Pt(CN)4 and KAu(CN)2 derivatives. The mean figure of merit mean value of m, for the 1106 reflexions used was 0.70. Difference Fourier syntheses for data collected from crystals soaked in platinum D-methionine and in 6-(4-hydroxy-3,5-di-iodobenzamido)penicilloic acid revealed the likely position of the active site of the enzyme.
The reaction of cow beta-lactoglobulin with Au(CN)-4 is shown to label the free sulphydryl group on the protein by a reductive mechanism yielding a stable complex. High salt concentrations are shown to affect the pH-dependent conformational transition (N in equilibrium R yields S). It is hoped that Au(CN)-4 may be of more widespread use as a specific heavy-atom label for the isomorphous replacement method.