Biomedical subjects

L Szilák

Publications and source records attributed to L Szilák.

9 recordsLinked to original sources

PubMed1997

Leucine is the most stabilizing aliphatic amino acid in the d position of a dimeric leucine zipper coiled coil.

The energetic contribution of seven amino acids in the d position of a dimeric leucine zipper coiled coil structure was measured by determining the thermal stability. The d position contains the conserved leucines found in the leucine zipper. We used a natural bZIP protein as our host-guest system that remains dimeric when a single d position is mutated. We have determined the thermal stability, monitored by circular dichroism, of 14 proteins which indicate that alanine is 4.6 kcal mol-1 per residue less stabilizing than leucine. The similarly sized amino acid isoleucine is 2.9 kcal mol-1 per residue less stabilizing than leucine, suggesting that leucine is well-packed. Model building indicates that the beta-branched amino acids isoleucine and valine in the d position produced interhelical clashes between the Cgamma2 methyl groups when placed in the favored rotamer conformation. The stabilization by leucine in different d positions is context-dependent; it varies by over 2 kcal mol-1 in the two positions examined. The order of stabilization is L, M, I, V, C, A, and S. Cysteine in the d position can form a disulfide bond which stabilizes the coiled coil.

Amino Acid Sequence↗

PubMed1997

Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.

Amino Acid Sequence↗

PubMed1997

Phosphorylation destabilizes alpha-helices.

Phosphorylation of threonine destabilizes the leucine zipper of a bZIP protein by 4.6 kcal mol-1 dimer-1, which reduces DNA binding 100-fold. This decrease in stability reflects the low alpha-helix forming propensity of a phosphorylated threonine.

Amino Acid Sequence↗

PubMed1995

Self-methylation of the M.BspRI methyltransferase.

In the absence of DNA substrate, the DNA methyltransferase (MTase) M.BspRI can methylate itself using the methyl donor S-adenosyl-L-methionine (AdoMet). The methyl group is transferred to two Cys residues of the MTase.

Amino Acid Sequence↗

PubMed1994

Self-methylation of BspRI DNA-methyltransferase.

The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA. Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation. Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents. Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with [methyl-3H]AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine. One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.

Amino Acid Sequence↗

PubMed1993

Kinetic characterization of the EcaI methyltransferase.

A kinetic analysis of the EcaI adenine-N6-specific methyltransferase (MTase) is presented. The enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the adenine of the GGTNACC sequence with a random rapid-equilibrium mechanism. Experiments with a synthetic, 14-bp DNA substrate suggest that recognition of the specific site of DNA occurs after the binding of AdoMet. Proton concentration does not affect the dissociation constant of AdoMet while Vm and the dissociation constant of DNA show a maximum around pH 8. Increasing the amount of S-adenosyl-L-homocysteine decreases the inhibitory effect of methylated DNA which proves the active role of AdoMet in site recognition. Experiments with hemimethylated DNA show that the methylase binds the double-stranded DNA asymmetrically.

Base Sequence↗

PubMed1992

Purification and biochemical characterization of the EcaI DNA methyltransferase.

The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25 degrees C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The Km was 170 nM for DNA and 1.8 microM for the methyl donor S-adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM). The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 microM). The S-adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.

Adenosine↗

PubMed1991

Complementation by detached parts of GGCC-specific DNA methyltransferases.

Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.BspRI can complement each other resulting in specific, in vivo methylation of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecies complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other.

Base Sequence↗

PubMed1990

Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes.

The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E. coli. The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease. No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease. The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases. In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC). M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence. The Sau96I endonuclease appears to act as a monomer.

Amino Acid Sequence↗