摘要:

AbstractThe hematopoietic growth factors are a family of glycoproteins involved in the production of blood cells from their bone marrow precursors and in the activation of mature blood cells. Much has been learned about the structural features of these molecules responsible for their characteristic biological activities. Most studies have been based upon mutagenesis strategies of intact polypeptides and on epitope mapping of informative monoclonal antibodies to the growth factors. A more limited amount of physical data is available. This review will summarize these findings, highlight the growing body of evidence suggesting that many of these proteins share common evolutionary origins and structural elements, and hopefully point to the directions being taken for further investigations of these scientifically informative and clinically useful group of proteins.

ISSN:0887-3585    

DOI:10.1002/prot.340120102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe molecular structure of interleukin‐1β, a hormone‐like cytokine with roles in several disease processes, has been determined at 2.0 Å resolution and refined to a crystallographicR‐factor of 0.19. The frame‐work of this molecule consists of 12 antiparallel β‐strands exhibiting pseudo‐3‐fold symmetry. Six of the strands make up a β‐barrel with polar residues concentrated at either end. Analysis of the three‐dimensional structure, together with results from site‐directed mutagenesis and biochemical and immunological studies, suggest that the core of the β‐barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL‐1

ISSN:0887-3585    

DOI:10.1002/prot.340120103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractGlycosylated interleukin‐2 (glyIL‐2) has been crystallized in two crystal forms, and unglycosylated interleukin‐2 (uIL‐2) has been crystallized in three forms. The glycosylated form of the human recombinant IL‐2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space groupP6222 or its enantiomorph. The crystals diffract to 2.8 Å and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0–5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL‐2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5–5.6 in the monoclinic space groupP21, and less frequently in the orthorhombic space groupP212121from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross‐seeding uIL‐2 with seeds from hexagonal crystals of glyIL‐2 promotes nucleation of trigonal crystals of unglycosylated IL‐2. These trigonal crystals belong to the space groupP3121 or its enantiomorph, with similar cell dimensions to the gl

ISSN:0887-3585    

DOI:10.1002/prot.340120104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA computer algorithm, CLIX, capable of searching a crystallographic database of small molecules for candidates which have both steric and chemical likelihood of binding a protein of known three‐dimensional structure is presented. The algorithm is a significant advance over previous strategies which consider solely steric or chemical requirements for binding. The algorithm is shown to be capable of predicting the correct binding geometry of sialic acid to a mutant influenzavirus hemagglutinin and of proposing a number of potential new ligands to this protei

ISSN:0887-3585    

DOI:10.1002/prot.340120105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA recombinant 19‐kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified fromE. coliinclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full‐length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24‐nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full‐length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19‐kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50∼60 μM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19‐kDa collagenase fragment is a multistep process stabili

ISSN:0887-3585    

DOI:10.1002/prot.340120106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn order to resolve whether gramicidin A channels are formed by right‐ or left‐handed β‐helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A−, to test whether it forms channels that have the same handedness as channels formed by gramicidin M−(F. Heitz et al., Biophys. J. 40:87–89, 1982). In gramicidin M−the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therfore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M−in dimyristoylphosphatidylcholine vesicles is consistent with a left‐handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149–160, 1986), and the CD spectra of gramicidins A and A−are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A−and M−channels are structurally equivalent, while gramicidin A and A−channels are nonequivalent, being of opposite helix sense. Gramicidin A−channels are therefore left‐handed, and natural gramicidin A channels in phospholipid bilayers are

ISSN:0887-3585    

DOI:10.1002/prot.340120107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin‐c, grown from three different calcium concentrations. It appeared that the B‐factors of the thermitase eglin complex obtained at 100 mM CaCl2and elucidated at 2.0 Å resolution are remarkably similar to those of the 1.4 Å native structure: the main chain atoms have an rms difference of only 2.3 Å2; for all atoms this difference is 4.6 Å2. The rms positional differences between these two structures of thermitase are 0.31 Å for the main chain atoms and 0.58 Å for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X‐ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect.A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2thermitase:eglin complex have an rms deviation of 0.13 Å for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 Å in Cαpositions. These are probably due to crystal packing effects.The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 Å. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition

ISSN:0887-3585    

DOI:10.1002/prot.340120108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe three‐dimensional crystal structure of the NAD+‐linked glutamate dehydrogenase fromClostridium symbiosumhas been solved to 1.96 Å resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographicRfactor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self‐assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for thesynconformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2‐oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalyt

ISSN:0887-3585    

DOI:10.1002/prot.340120109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe class I major histocompatibility (MHC) antigen HLA‐B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG‐2. Detergent‐soluble HLA‐B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water‐soluble extracellular fragments were obtained in hanging drops by the vapor‐diffusion method. The crystals are triclinic, space groupP1, with unit cell dimensionsa= 45.9 Å,b= 71.0 Å,c= 83.7 Å, α = 79.4°, β = 88.5°, γ = 89.9°, and diffract bey

ISSN:0887-3585    

DOI:10.1002/prot.340120110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractHexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time‐resolved X‐ray crystallographic experiments. Substrates used in this study were hexa‐N‐acetylglucosamine (hexa‐GlcNAc) and a modified analogue of hexa‐GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled β‐N‐acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X‐ray experiment. Unmodified hexa‐GlcNAc is hydrolyzed into di‐, tri‐, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (± 1.0) × 10−7mmol/min/mg pr

ISSN:0887-3585    

DOI:10.1002/prot.340120111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY