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J J Stezowski

Publications and source records attributed to J J Stezowski.

At least 19 recordsLinked to original sources
PubMed1996

The surface of beta-sheet proteins contains amphiphilic regions which may provide clues about protein folding.

A major bottleneck in the field of biochemistry is our limited understanding of the processes by which a protein folds into its native conformation. Much of the work on this issue has focused on the conserved core of the folded protein. However, one might imagine that a ubiquitous motif for unaided folding or for the recognition of chaperones may involve regions on the surface of the native structure. We explore this possibility by an analysis of the spatial distribution of regions with amphiphilic alpha-helical potential on the surface of beta-sheet proteins. All proteins, including beta-sheet proteins, contain regions with amphiphilic alpha-helical potential. That is, any alpha-helix formed by that region would be amphiphilic, having both hydrophobic and hydrophilic surfaces. In the three-dimensional structure of all beta-sheet proteins analyzed, we have found a distinct pattern in the spatial distribution of sequences with amphiphilic alpha-helical potential. The amphiphilic regions occur in ring shaped clusters approximately 20 to 30 A in diameter on the surface of the protein. In addition, these regions have a strong preference for positively charged amino acids and a lower preference for residues not favorable to alpha-helix formation. Although the purpose of these amphiphilic regions which are not associated with naturally occurring alpha-helix is unknown, they may play a critical role in highly conserved processes such as protein folding.

Amino Acids↗
PubMed1993

Crystallization and preliminary X-ray data of chloromuconate cycloisomerase from Alcaligenes eutrophus JMP134 (pJP4).

The pJP4-encoded chloromuconate cycloisomerase, an enzyme of the 2,4-dichlorophenoxy-acetate degradation pathway, was purified from cell-free extracts of Alcaligenes eutrophus JMP134 with a revised procedure. Tetragonal bipyramidal crystals were grown and characterized with respect to their X-ray diffraction properties. They were assigned to the space group I4, with cell dimensions of a = b = 111.9 A, c = 148.5 A. The crystals scattered to approximately 3 A resolution.

Alcaligenes↗
PubMed1989

Preliminary X-ray crystallographic study of malate dehydrogenases from the thermoacidophilic Archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius.

Malate dehydrogenases from the thermoacidophilic Archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius have been crystallized and characterized by X-ray diffraction measurements. Crystals of the enzyme from T. acidophilum display space-group symmetry P2(1), a = 63 A, b = 135 A, c = 83 A and beta = 105 degrees; they scattered to approximately 4 A resolution. Two crystal modifications of malate dehydrogenase from S. acidocaldarius were characterized; one displayed trigonal symmetry corresponding to space groups P321, P3(1)21 or P3(2)21 with lattice parameters a = 151 A and c = 248 A and with resolution approximately to 5 A, whereas the other modification displayed space group symmetry I23 or I2(1)3 with lattice parameters a = 129 A and approximately 4.5 A resolution.

Archaea↗
PubMed1987

Preparation and characterization in solution of oligonucleotides alkylated by activated carcinogenic polycyclic aromatic hydrocarbons.

The effects of aralkylation of selected oligonucleotides by a bulky chemical carcinogen, 7,12-dimethylbenz(a)anthracene (after activation) have been studied. The aralkylation involves the base adenine, designated A* at the modification site, in the center of synthetic heptameric, nonameric and pentadecameric oligonucleotides; complementary strands lacking any modification were also synthesized. The products were studied by UV melting curves and CD spectral techniques. Duplex formation was modified by such aralkylation of a central base in the oligomers. The extent of duplex formation was found to depend on chain length as follows: no evidence was found for duplex formation of the heptamer d(GTCA*GAC) + d(GTCTGAC); the nonamer, d(GTGCA*ATCC) + d(GGATTGCAC), appears to form a duplex at high salt concentrations and reduced temperature; the pentadecamer, d(CCGCT-GCGA*TCCGGC) + d(GCCGGATCGCAGCGG), forms a duplex at low salt concentration and room temperature, but its melting temperature is lower than that of the nonalkylated parent system. CD-spectra for the duplexes formed by the nonamer or pentadecamer are indicative of a right-handed helical conformations. On phosphordiesterase digestion it appears that the aralkylated adenine and the base on its 5'-side act as "stops" for enzymatic digestion from either direction. We suggest, from model building, that this inhibition of phosphodiesterase activity is the result of the steric bulk and disposition of the polycyclic aromatic hydrocarbon. We further suggest that unusual base pairing (mismatching), such as A...A, which would lead to an AT transversion, may be favored by the bulkiness of the aromatic group.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1987

Crystalline NAD/NADP-dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

Malate dehydrogenase from Sulfolobus acidocaldarius has been purified 240-fold to apparent electrophoretic homogeneity. The enzyme shows a specific activity of 277 U/mg and crystallizes readily. The relative molecular mass of the native enzyme is estimated as 128,500 by ultracentrifugation. After cross-linking a relative molecular mass of 134,000 is found by sodium dodecyl sulfate gel electrophoresis. Malate dehydrogenase from S. acidocaldarius is composed of four subunits of identical size with a relative molecular mass of 34,000. Active-enzyme sedimentation in the analytical ultracentrifuge indicates that the tetramer is the catalytically active species. Kinetic studies in the direction of oxaloacetate reduction showed a Km for NADH of 4.1 microM and a Km for oxaloacetate of 52 microM. Oxaloacetate exhibits substrate inhibition at higher concentrations, L-malate, NAD and NADP were found to be product inhibitors. The enzymatic activity is inhibited by 2-oxoglutarate but not by the adenosine nucleotides AMP, ADP and ATP. Only low activity is detected in the direction of malate oxidation. Malate dehydrogenase from S. acidocaldarius utilizes both NADH and NADPH to reduce oxaloacetate. The enzyme shows A-side stereospecificity for both nicotinamide dinucleotides.

Amino Acids↗
PubMed1986

Structure of pressinoic acid: the cyclic moiety of vasopressin.

Arginine vasopressin consists of a 20-membered, disulfide-linked macrocyclic ring system called pressinoic acid to which is attached a COOH-terminal tripeptide. The molecular conformation of pressinoic acid has been determined from single crystal x-ray diffraction data. The 20-membered macrocyclic ring, stabilized by two intramolecular hydrogen bonds, has a type I beta-bend centered on Gln4 and Asn5 and a highly distorted type II' bend centered on Phe3 and Gln4. In vasopressin the Asn5 side chain extends away from the macrocyclic ring system and hydrogen bonds to the terminal tripeptide, but in pressinoic acid the Asn5 side chain lies over the molecule and forms a strong hydrogen bond to the nitrogen of Tyr2. The absence of pressor activity in pressinoic acid may be a result of both the loss of the COOH-terminal tripeptide and the incorrect orientation of the Asn5 side chain. Whether this class of hormones has pressor or oxytocic activity is determined by the orientation of the Tyr2 side chain, that is, whether it is extended away from or over the ring system, respectively. In pressinoic acid, the Tyr2 side chain is in the expected "pressor conformation," that is, extended away from the ring system, and is stabilized through a hydrophobic interaction with the Phe3 side chain. Thus, the conformation of the pressinoic acid molecule partly explains the activity of vasopressin-like hormones.

Arginine Vasopressin↗
PubMed1986

Purification and properties of malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

Malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum is purified 50-fold to electrophoretic homogeneity. The purified enzyme crystallizes readily. Native malate dehydrogenase shows a relative molecular mass of 144 000. It is a tetramer of identical subunits with a relative molecular mass of 36 600. Malate dehydrogenase from Thermoplasma uses both NADH and NADPH as coenzyme to reduce oxaloacetate. The enzyme shows A-side (pro-R) stereospecificity for both coenzymes. The pH optimum for the reduction of oxaloacetate in the presence of NADH is found to be at pH 8.1. At pH 7.4 the Km value for oxaloacetate is found to be 5.6 microM while for NADH a value of 11.7 microM is found. The homogeneous enzyme shows a turnover number of kcat = 108 s-1.

Crystallization↗
PubMed1985

Archaebacterial malate dehydrogenases. The enzymes from the thermoacidophilic organisms Sulfolobus acidocaldarius and Thermoplasma acidophilum show A-side stereospecificity for NAD+.

The stereoselective transfer of hydrogen from NADH to oxaloacetate catalysed by malate dehydrogenases (EC 1.1.1.37) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius and Thermoplasma acidophilum was studied by the p.m.r. method described by Zhou & Wong [(1981) J. Biochem. Biophys. Methods 4, 329-338]. Both enzymes are A-side (pro-R) stereospecific for NADH.

Archaea↗
PubMed1985

Conformational properties of central nervous system active thyrotropin releasing hormone analogues: probing structure-activity relationships at the molecular level.

Crystal structure determinations have been carried out for the following seven thyrotropin releasing hormone analogues: I, (3R,6R)-6-methyl-5-oxothiomorpholin-3-ylcarbonyl-L- histidinyl-L-proline amide; II, (3R,6S)-6-methyl-5-oxothiomorpholin-3-ylcarbonyl-L-histid inyl-L-proline amide; III, (4R)-2-oxothiazolidin-4-ylcarbonyl-D-histidinyl-L-prol ine amide; IV, 5-ethylorotyl-L-histidinyl-L-proline amide; V, 5-n-propylorotyl-L-histidinyl-L-proline amide; VI, 5-bromoorotyl-L-histidinyl-L-proline amide; and VII, Phe2-TRH. A surprising degree of conformational similarity has been observed for the peptide backbone. All peptide bonds are found to be trans. A composite hydrogen-bonding environment has been constructed for the TRH analogue system and examined for its inference with respect to receptor binding. A comparison of the conformations of these analogues with those displayed by Leu5-enkephalin has also been made, and unexpected similarities have been revealed.

Crystallization↗
PubMed1985

Molecular level studies with anthracyclines.

There is steadily growing interest in the characterization of chemotherapeutic agents at the molecular level. Such characterization ideally involves the elucidation of structural properties such as configuration, conformation and tautomeric or mesomeric form, establishes the effect of chemical substitution on structural properties and provides insight into probable intermolecular interactions that are environmentally dependent. The interpretation of these data can be important for the design of new chemotherapeutic agents. The authors are presently investigating the anthracyclines, particularly members of the daunomycin and nogalamycin families, in an effort to better understand their behaviour at the molecular level. These studies have drawn upon analysis of single crystal structures determined with X-ray diffraction data and solution studies employing ultraviolet and circular dichroism spectroscopies. Crystal structures have been determined for 7-con-O-methylnogarol, 7-deoxynogarol, 9-deacetyldaunomycin, adriamycin-14-valerate (AD-48), and N-trifluoroacetyladriamycin-14-valerate (AD-32). Solution studies have concentrated on the study of the interaction of selected anthracyclines with oligonucleotides of known sequence. The oligonucleotides used were: d-(5GTCATGAC), d-(5GTCGCGAC) and the non-self complementary pair d-(5GTCGTCA) and d-(5TGACGAC).

Antibiotics, Antineoplastic↗
PubMed1984

Intercalation model for DNA-cross linking in a 1-nitro-9-aminoacridine derivative, an analog of the antitumor agent "ledakrin" (nitracrine).

Ledakrin (nitracrine), C-283, is a 1-nitro-9-aminoacridine derivative that is used in Poland as an antitumor agent. In order to investigate the basis of the activity of this compound the structure of another analog, [9-(3-dimethyl-1-methylpropylimino)-1-nitro-9,10-dihydroacridin e], C-829, that has similar activity, was determined by X-ray crystallographic techniques and was compared with that of ledakrin, already reported in the literature. In both molecules the proximity of the 1-nitro to the substituted 9-aminoacridine group causes extensive distortions. These compounds are believed to act, after metabolic "activation", by cross-linking DNA. Such cross-linking does not occur in the absence of the 1-nitro group or if the nitro group is moved to the 2-, 3- or 4-position. Computer-assisted model-building has been used to test possible intercalative models. It has shown that functional groups on C-829 or C-283 are, when the acridine portion of the molecule is intercalated as in a proflavine dinucleoside phosphate complex, in positions suitable for DNA cross-linking by activated 1-nitro-9-aminoacridine derivatives.

Aminoacridines↗
PubMed1984

Stereochemical properties of nucleosides alkylated by activated carcinogens.

An initial stage in the mechanism of chemical carcinogenesis by "activated" carcinogenic polycyclic aromatic hydrocarbons is believed to involve alkylation of DNA. However, very high (atomic) resolution studies of alkylated DNA are not technically feasible at this time, and therefore the detailed, high-resolution three-dimensional structures of portions of alkylated DNA have been determined. The initial phase of this study (reported here) has involved the preparation of a series of adenosines and 2'-deoxyadenosines substituted at N6 by related aralkyls of differing carcinogenic potential. We report here the crystal structure determinations of four of these compounds: Compound 1, N6-(anthracenyl-9-methyl)adenosine; Compound 2, N6-(10-methyl-anthracenyl-9-methyl)adenosine; Compound 3, N6-[12-methyl-benz(a)anthracenyl-7-methyl]adenosine; and Compound 5, N6-(10-methylanthracenyl-9-methyl)-2'-deoxyadenosine. Results are compared with those for a previously published analysis Compound 6, N6-[12-methylbenz(a)anthracenyl-7-methyl]-2'-deoxyadenosine. Several results of structural interest have emerged. All five compounds have the syn-conformational relationship between the sugar (ribose or 2'-deoxyribose) and the base (adenine), in contrast to the anti arrangement in B-DNA and in nonalkylated nucleosides. In four of the five compounds, there is an intramolecular hydrogen bond between the 5'-hydroxyl group and adenine. However, in the fifth molecule, this hydrogen bond is not found, and yet the conformation is syn. This indicates that formation of this internal hydrogen bond is not a prerequisite for the adoption of the syn-conformation. In general, the overall conformations of all five compounds are similar, the base lying approximately perpendicular to the polycyclic aromatic ring system. The packing of molecules in the unit cell is also of interest because it consists of alternations of adenine and polycyclic aromatic ring systems in columns through the crystal, indicating that this may serve as a model for the interaction with DNA. The oxygen atom of the sugar ring points towards the hydrocarbon ring system of another molecule. It is premature at this stage of our study to speculate as to the effects of alkylation on the conformational properties of either RNA or DNA. The only comment that appears justified is that the propensity of these adducts to adopt the syn-conformation may be indicative of a preference of alkylated DNA for the Z-conformation (even if the form that is initially attacked is B-DNA).(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗