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A Galat

Publications and source records attributed to A Galat.

34 records · Page 2Linked to original sources
PubMed1991

Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus.

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (Kd = 0.4 nM) and rapamycin (Kd = 0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFV- LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino acid sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other posttranslational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaert et al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent beta-strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and overexpression of the human FK506-binding protein FKBP.

The potent immunosuppressive agent FK506 is highly effective in preventing organ transplant rejection in humans. Like cyclosporin A, FK506 inhibits the transcription of early T-cell activation genes, apparently by modulating the activity of transcriptional regulators such as nuclear factor of activated T cells. A remarkable finding is that the predominant binding proteins (immunophilins) for cyclosporin A and FK506, cyclophilin and FKBP respectively, are peptidyl-prolyl-cis-trans-isomerases that are potently and selectively inhibited by their respective ligands. Here we report the complementary DNA and derived amino-acid sequences of human FKBP from Jurkat cells and also the efficient overexpression in Escherichia coli of fully active, recombinant human FKBP. The human FKBP cDNA sequence shows significant similarity to an open reading frame in the Neisseria meningitidis genome.

Amino Acid Isomerases↗
PubMed1990

Inhibition of FKBP rotamase activity by immunosuppressant FK506: twisted amide surrogate.

The immunosuppressive agents cyclosporin A and FK506 inhibit the transcription of early T cell activation genes. The binding proteins for cyclosporin A and FK506, cyclophilin and FKBP, respectively, are peptidyl-prolyl-cis-trans isomerases, or rotamases. One proposed mechanism for rotamase catalysis by cyclophilin involves a tetrahedral adduct of an amide carbonyl and an enzyme-bound nucleophile. The potent FKBP rotamase inhibitor FK506 has a highly electrophilic carbonyl that is adjacent to an acyl-pipicolinyl (homoprolyl) amide bond. Such a functional group would be expected to form a stabilized, enzyme-bound tetrahedral adduct. Spectroscopic and chemical evidence reveals that the drug interacts noncovalently with its receptor, suggesting that the alpha-keto amid of FK506 serves as a surrogate for the twisted amide of a bound peptide substrate.

Amino Acid Isomerases↗
PubMed1990

Molecular models of neocarzinostatin damage of DNA: analysis of sequence dependence in 5'GAGCG:5'CGCTC.

Model building and molecular mechanics and dynamics calculations have been performed on a number of complexes of the post-activated form of the neocarzinostatin chromophore (NCS) with the B-DNA oligomer 5'GAGCG:5'CGCTC. Stable structures with the naphthoic acid moiety intercalated at all base pairs can be constructed. The observed bistranded lesions consisting of an abasic site at the Cyt residue in AGC and a direct break at the Thy residue on the complementary strand can be explained by assuming that NCS in the (R,R) form intercalates between the Ade2-Thy9/Gua3-Cyt8 base step with its 'diradical' core oriented towards the 3'-end of the (+) strand. Sites at C5', C4' and C1' in the minor groove are within a short enough distance from the two radical centers on NCS to permit hydrogen atom abstraction and the formation of the bistranded lesions. Strand cleavage at Thy9 may occur as a single lesion if NCS is intercalated into the Gua3-Cyt8/Cyt4-Gua7 base step with its active core towards the 3'-end of the (-) strand. The results are analyzed, and the utility and limitations of this type of model building are discussed.

Antibiotics, Antineoplastic↗
PubMed1990

A note on sequence-dependence of DNA structure.

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.

Base Sequence↗
PubMed1990

A note on graphing helical parameters of dynamics structure of DNA.

A graphical procedure for analysis of helical parameters in dynamic structure of DNA is described. The performance of the procedure is illustrated by analysis of a 20 ps dynamics simulation of the non-self-complementary ninemer, 5'CAAACAGGA:5'TCCTGTTTG, which is a part of DNA from lacI gene. The dynamics trajectory of the duplex shows sequence-dependent fluctuations of helical parameters.

Base Sequence↗
PubMed1989

A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase.

The structurally novel macrolide FK506 (refs 1,2) has recently been demonstrated to have potent immunosuppressive activity at concentrations several hundredfold lower than cyclosporin A (CsA). Cyclosporin A, a cyclic peptide, has found widespread clinical use in the prevention of graft rejection following bone marrow and organ transplantation. The mechanisms of immunosuppression mediated by FK506 and CsA appear to be remarkably similar, suggesting that these unrelated structures act on a common receptor or on similar molecular targets, perhaps the CsA receptor, cyclophilin, which has recently been shown by Fischer et al. and Takahashi et al. to have cis-trans peptidyl-prolyl isomerase activity. We have prepared an FK506 affinity matrix and purified a binding protein for FK506 from bovine thymus and from human spleen. This FK506-binding protein (FKBP) has a relative molecular mass (Mr) of approximately 14,000(14K), a pI of 8.8-8.9, and does not cross-react with antisera against cyclophilin. The first 40 N-terminal residues of the bovine and 16 residues of the human FKBP were determined; the 16-residue fragments are identical to each other and unrelated to any known sequences. This protein catalyses the cis-trans isomerization of the proline amide in a tetrapeptide substrate and FK506 inhibits the action of this new isomerase. The FKBP and cyclophilin appear to be members of an emerging class of novel proteins that regulate T cell activation and other metabolic processes, perhaps by the recognition (and possibly the isomerization) of proline-containing epitopes in target proteins.

Amino Acid Isomerases↗
PubMed1989

A procedure for analysis of densitometric spectra.

Computer-aided quantitative analysis of densitometric spectra is presented. The densitometric spectra are decomposed into component bands using the Powell and Marquardt minimizers. Several different functions for the component bands are utilized. It is shown that the densitometric spectra can be decomposed into component bands with high accuracy only if the proper shapes of the bands are chosen. The method described was used for quantitative analysis of densitometric spectra of DNA cleaved by neocarzinostatin. The procedure is general and can be applied to analyses of autoradiographic films and to direct scans of electrophoretic gels. It is shown that densitometric spectra which show highly overlapped bands are well approximated by asymmetric Cauchy and Gauss functions. Well separated densitometric bands which have substantial asymmetry can be fitted to more sophisticated shapes formed by combinations of symmetric and asymmetric Cauchy and Gauss functions. High accuracy fitting to asymmetric densitometric curves may only be achieved using the cosine function introduced by Mignot and Rondot, J. Appl. Cryst. 1976, 9,460-465.

Base Sequence↗
PubMed1989

Analysis of dynamics trajectories of DNA and DNA-drug complexes.

A FORTRAN-77 program is described which was applied for analysis of optimized structures and computer-generated dynamics trajectories of DNA and DNA-drug complexes. The CORDAN program (coordinates analysis) also can be used for various manipulations of DNA-drug complexes, i.e. inversion of asymmetric sites or rebuilding the structure of the intercalator, among others. These procedures can find application in drug design. Analysis of dynamics trajectory of neocarzinostatin antibiotic (NCS) intercalated to the A-DNA form of 5'GGATGGGAG:5'CTCCCATCC is presented. The procedures described can be used for detailed analysis of dynamics structures of DNA and their complexes with intercalating drugs.

Base Sequence↗
PubMed1989

CORGEN: a FORTRAN-77 generator of standard and non-standard DNA helices from the sequence.

An analytical procedure CORGEN generates a variety of DNA double-stranded structures from user-supplied sequence using a nucleic acid database incorporated into a standard FORTRAN-77 program. Alternatively, the cylindrical polar coordinates of DNA components may be supplied from the external table. An algorithm that performs intercalation sites in DNA is described. This procedure can be used to generate complexes of antibiotics with DNA. Non-standard DNA structures can be built by alternating the global helical twist and global helical rise in the regular DNA helix. The procedures described can be used for computer generation of a variety of non-standard DNA structures which can be subjected to molecular mechanics and dynamics simulations.

Base Sequence↗
PubMed1988

Interaction of riboflavin binding protein with riboflavin, quinacrine, chlorpromazine and daunomycin.

1. circular dichroism and fluorescence measurements were used to study the reversible unfolding of riboflavin-riboflavin binding protein complex. Both methods showed that the complex was unfolded according to the two-state model. 2. The results suggest that riboflavin was strongly bound in the hydrophobic cleft of the protein and that it could not be dislodged by TFE, MeOH or SDS without major unfolding of the unique tertiary structure of the protein. 3. In addition, it has been also shown that quinacrine, chlorpromazine and daunomycin did not form stereospecific complexes with riboflavin binding protein.

Carrier Proteins↗
PubMed1987

Analysis of microdensitometric data in terms of probability of cleavage of DNA.

Computer-aided analysis of autoradiographic films of DNA fragments is presented. The Powell least-squares procedure is used for optimization of parameters for components of complex densitometric curves. Since each densitometric spectrum may be divided for several non-overlapped blocks of bands, there is no upper limit on the number of parameters which must be optimized. Eight shapes for the component bands are utilized: symmetric and asymmetric Gauss and Cauchy functions, direct, symmetric and asymmetric product of Gauss function and inverse of Cauchy function, and log-normal function. The probability of DNA cleavage is calculated with correction for multiple cuts. The methods presented was applied to detailed analysis of densitometric spectra of a 21-bp DNA restriction fragment and allowed for direct correlation between structural microheterogeneity of DNA and the resulting cutting pattern. This method should facilitate the analysis of densitometric data from antibiotic-induced cleavage of DNA and footprinting experiments.

Absorptiometry, Photon↗
PubMed1986

Computer-aided analysis of infrared, circular dichroism and absorption spectra.

Marquardt and Powell optimization methods without constraints on the optimized spectral parameters were employed for decomposition of complex i.r., c.d. and absorption spectra into component bands. The procedure resolved experimental spectra into eight component bands and it can be easily adjusted for a larger set of component bands. The CPU time required for achievement of satisfactory convergence of parameters for eight component bands is rather large even when using mainframe computers and therefore division of spectra into a few non-overlapped parts is advisable. The program also can be used for calculation of absorption, c.d. and difference spectra from formatted raw spectral data.

Algorithms↗
PubMed1981

Circular dichroism, Raman spectroscopy, and gel filtration of trapped folding intermediates of ribonuclease.

The intermediates of ribonuclease with one to four disulfide bonds trapped during refolding of the reduced protein have been compared to the fully reduced and native forms of the protein by gel filtration, circular dichroism, and Raman spectroscopy. Correctly refolded ribonuclease is indistinguishable from native protein, while a three-disulfide intermediate has a compact conformation which is very similar, but not identical. In contrast, all other intermediates with one to four disulfide bonds are qualitatively similar to fully reduced ribonuclease by their circular dichroism and Raman spectra, although the disulfide cross-links affect the dimensions of the polypeptide chain. The apparent absence of stable partially ordered structures in the initial disulfide intermediates implies that during refolding there are relatively few constraints on formation on disulfide bonds, although their formation is probably not entirely random. The stable conformation appears during refolding only when the three or four disulfide bonds capable of stabilizing a native-like conformation of the entire polypeptide chain occur simultaneously.

Chromatography, Gel↗
PubMedDate not supplied

Convergence of amino acid compositions of certain groups of protein aids in their identification on two-dimensional electrophoresis gels.

The amino acid composition (AAC) versus the protein identity (PI) method was used for establishment of the identities of proteins from bovine brain and kidneys which were prefractionated on a CM52 cation exchanger and by preparative flat-bed isoelectric focusing. Established identities of proteins whose AACs converge with those of other members of their proper superfamily are reliable. Groups of convergent AACs can be extracted from protein databases using the standard root-mean-square rule (Rmsd) with measures the difference between the AAC of chosen protein versus those in the database. Convergence of AACs of proteins is dependent on several factors such as the upper limit of Rmsd, the limits of variations of molecular mass (m) and isoelectric point (pI), the number of proteins with similar AACs present in protein databases, and the domain structure of proteins. AACs of many proteins remain unique if the Rmsd is maintained within 1.5-1.0 with m +/- 3kDA and pI +/- 4. Certain groups of multidomain proteins have quasi-unique AACs only if the Rmsd is restrained to a value within 1.0 and 0.7. Convergence of AACs of certain groups of proteins may indicate that a common biological function exists for some members of each group. The AAC-PI method may become an additional search tool for protein functions.

Amino Acid Isomerases↗