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Biomedical subjects

L Sawyer

Publications and source records attributed to L Sawyer.

At least 55 records · Page 3Linked to original sources
PubMed1994

Probing the retinol-binding site of bovine beta-lactoglobulin.

The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites. Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft. To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket. The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins. Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues. The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins. The blue shift indicates protonation of the Schiff base. The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol. These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.

Amino Acid Sequence↗
PubMed1994

Synthetic, structural and biological studies of the ubiquitin system: chemically synthesized and native ubiquitin fold into identical three-dimensional structures.

The solid-phase chemical synthesis of ubiquitin produced a molecule with physicochemical properties similar to those of the natural protein. We have grown crystals of this synthetic ubiquitin and performed an X-ray analysis at 1.8 A resolution in order to compare the synthetic protein with the known natural structure. The crystals were isomorphous with those of the natural protein, the R-factor between them being 7.1%. Difference Fourier analysis shows that the synthetic and natural structures are indistinguishable. The co-ordinates of the natural ubiquitin (1UBQ) were used as the starting point for restrained least-squares refinement (TNT program) against the synthetic X-ray data. The refinement converged to R = 16.5% and the resulting model did not change when refined against natural ubiquitin X-ray data (R = 18.7%). From both the refinement and featureless difference Fourier synthesis, we conclude that the synthetic and natural protein structures are identical. A short discussion about the uses of proteins with 'non-standard' amino acid residues is included.

Crystallography, X-Ray↗
PubMed1994

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis.

The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available. The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination. The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees. The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit.

Amino Acid Sequence↗
PubMed1994

Occupational asthma and the chemical properties of low molecular weight organic substances.

Various low molecular weight substances cause occupational asthma, and there is scope for studying their chemical properties in relation to their propensity to cause this condition. Twenty-nine organic substances (molecular weight < 250), with or without oxygen or nitrogen atoms, were compared to control hazardous substances obtained from the list of occupational exposure limits. There were significantly more reactive groups in the active chemicals than in the controls. Carbon to nitrogen double bonds were also significantly over-represented in the active chemicals. If confirmed in a fuller study, these data could yield useful information on the causation of occupational asthma and on the hazard assessment of novel chemical entities.

Air Pollutants↗
PubMed1994

Loss of red blood cell viability associated with limited thermal inactivation of extracellular HIV-1.

The effects of incubation at mildly elevated temperatures on HIV-1 inactivation and in vitro red blood cell properties were investigated. Red cells (55% Hct) were leukodepleted (3 log10) by filtration, maintained at 45 or 47 degrees C for 4 or 8 h, and then stored at 4 degrees C. Hemolysis was twice that of controls after 42-day storage for samples treated for 4 h at 45 degrees C, and five times larger for samples heated at 47 degrees C. There was also a significant increase in the rate of potassium loss, an early decrease in ATP levels, and an initial drop in pH for samples treated at either temperature. Larger differences were observed for samples exposed to these elevated temperatures for 8 h. Osmotic deformability curves obtained by ektacytometry showed dramatic decreases in red cell deformability at both temperatures and for both time periods. HIV-1 inactivation in red cells treated at 45 degrees C (approximately 0.25 log10/h) was considerably less than that obtained in tissue culture medium (1-2 log10/h). Since the decrease in red cell deformability is likely to indicate reduced red cell function and survival, and the rate of HIV-1 inactivation is low, mild heat treatment is not an adequate process for viral inactivation of red cell products.

Blood↗
PubMed1994

Neutralizing antibody responses to autologous and heterologous isolates of human immunodeficiency virus.

Although laboratory-adapted strains of human immunodeficiency virus (HIV) are generally highly sensitive to neutralization by HIV-positive patient sera, we have found a more complex pattern of cross-neutralization and neutralization resistance among low-passage clinical isolates. These HIV isolates, like many other lentiviruses, resisted neutralization by the patient's own (autologous) antibodies. We assessed the degree of antigenic relatedness between different patient isolates of HIV through cross-neutralization with heterologous sera and virus isolates. Complicated patterns emerged, with variation in breadth of neutralization among individual plasmas and variation in frequency of neutralization among isolates. In longitudinal studies of individuals, we found that some but not all such patients develop a neutralizing response that "catches up" with their earlier isolates after a lag period. Taken together, these data suggest that an individual's immune response broadens with time because of cumulative exposure to multiple antigenic variants that arise throughout HIV disease.

Cells, Cultured↗
PubMed1993

The secondary structure of milk proteins and their biological function.

A brief overview is given of the methods of determining and predicting secondary structure in proteins. The secondary structures of the milk serum proteins, lactoferrin, alpha-lactalbumin, lysozyme, and beta-lactoglobulin, as determined by x-ray crystallography, are compared with the results of a joint prediction method. This comparison evaluates critically the degree of success achieved and helps define what can reasonably be expected from a prediction in the absence of a known structure. The value of supplementary information from spectroscopic methods and the use of templates and sequence information from related proteins in improving the confidence of predictions are illustrated. One point that emerges is the general overprediction of helix content by the joint prediction method such that, for an all-beta protein such as beta-lactoglobulin, the method of Garnier, Osguthorpe, and Robson, applied with the correctly selected decision constants, provides a somewhat better approach. Secondary structure of the caseins can be predicted with less confidence than for globular proteins, and the results should be interpreted as evidence of a propensity to form transient secondary structures of the indicated type in view of the generally open and flexible conformation of caseins in solution.

Amino Acid Sequence↗
PubMed1992

Crystallization of a type I 3-dehydroquinase from Salmonella typhi.

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.

Bacterial Proteins↗
PubMed1992

Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila.

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.

Alcohol Dehydrogenase↗
PubMed1992

Transglutaminase catalyses the modification of glutamine side chains in the C-terminal region of bovine beta-lactoglobulin.

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.

Amino Acid Sequence↗
PubMed1991

Selective modification by transglutaminase of a glutamine side chain in the hinge region of the histidine-388----glutamine mutant of yeast phosphoglycerate kinase.

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.

Amino Acid Sequence↗
PubMed1991

Secondary structures of narcissus mosaic virus coat protein.

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.

Capsid↗
PubMed1990

Structure of the thromboxane receptor antagonist EP 092.

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].

Molecular Conformation↗
PubMed1988

The predicted secondary structures of class I fructose-bisphosphate aldolases.

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].

Animals↗