ABSTRACT
BACKGROUND: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. METHODS: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. RESULTS: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. CONCLUSIONS: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. GENERAL SIGNIFICANCE: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.
Subject(s)
DNA, Bacterial/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Fimbriae Proteins/metabolism , Polyethylene Glycols/metabolism , DNA, Bacterial/chemistry , Escherichia coli/chemistry , Magnetic Phenomena , Nucleic Acid ConformationABSTRACT
Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 microM(-1) min(-1).
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Liver/enzymology , Transglutaminases/antagonists & inhibitors , Animals , Biochemistry/methods , Drug Design , Drug Evaluation, Preclinical , Guinea Pigs , Kinetics , Peptides/chemistry , Structure-Activity RelationshipABSTRACT
Herein, we report the results of irreversible inhibition of guinea pig liver transglutaminase (TGase) by a series of 24 novel dipeptides containing either an alpha,beta-unsaturated amide or an epoxide functional group. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 421 x 10(3) and 3000 x 10(3)M(-1)min(-1).
Subject(s)
Amides/pharmacology , Enzyme Inhibitors/pharmacology , Epoxy Compounds/pharmacology , Liver/drug effects , Transglutaminases/antagonists & inhibitors , Amides/chemistry , Animals , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Epoxy Compounds/chemistry , Guinea Pigs , Kinetics , Liver/enzymology , Magnetic Resonance SpectroscopyABSTRACT
Herein we report the development of a direct and continuous spectrophotometric method for determining transglutaminase (TGase) activity by using N,N-dimethyl-1,4-phenylenediamine (DMPDA) as a gamma-glutamyl acceptor substrate and carbobenzyloxy-l-glutamylglycine (Z-Gln-Gly) as a typical peptide gamma-glutamyl donor substrate. The transamidation activity of TGase can thus be followed by monitoring the increase of absorbance of the resulting anilide product at 278 nm. The extinction coefficient of the authentic, independently synthesized anilide was determined to be epsilon = 8940 +/- 55 M(-1) cm(-1). Using this assay, we determined the apparent K(M) of DMPDA to be 0.25 mM, which compares favorably to the apparent K(M) values determined for other acceptor substrates under conditions where Z-Gln-Gly is also used as the donor substrate, such as N-acetyl-l-lysine methyl ester (9.6 mM) and methylamine (13.1 mM). Finally, the sensitivity of this assay technique was established through the measurement of irreversible inhibition constants for iodoacetamide, determined to be K(I) = 75 +/- 11 nM and k(inact) = (120 +/- 1) x 10(5) M(-1) min(-1).