Biomedical subjects

T E Strzelecka

Publications and source records attributed to T E Strzelecka.

5 recordsLinked to original sources

PubMed1995

The solution structure of the Mu Ner protein reveals a helix-turn-helix DNA recognition motif.

BACKGROUND: The Mu Ner protein is a small (74 amino acids), basic, DNA-binding protein found in phage Mu. It belongs to a class of proteins, the cro and repressor proteins, that regulate the switch from the lysogenic to the lytic state of the phage life cycle. There is no significant sequence identity between Mu Ner and the cro proteins of other phages, despite their functional similarity. In addition, there is no significant sequence identity with any other DNA-binding proteins, with the exception of Ner from the related phage D108 and the Nlp protein of Escherichia coli. As the tertiary structures of Mu Ner and these two related proteins are unknown, it is clear that a three-dimensional (3D) structure of Mu Ner is essential in order to gain insight into its mode of DNA binding. RESULTS: The 3D solution structure of Mu Ner has been solved by 3D and 4D heteronuclear magnetic resonance spectroscopy. The structure consists of five alpha helices, two of which comprise a helix-turn-helix (HTH) motif. Analysis of line broadening and disappearance of crosspeaks in a 1H-15N correlation spectrum of the Mu Ner-DNA complex suggests that residues in these two helices are most likely to be in contact with the DNA. CONCLUSIONS: Like the functionally analogous cro proteins from phages lambda and 434, the Mu Ner protein possesses a HTH DNA recognition motif. The Ner protein from phage D108 and the Nlp protein from E. coli are likely to have very similar tertiary structures due to high amino-acid-sequence identity with Mu Ner.

Amino Acid Sequence↗

PubMed1995

DNA binding specificity of the Mu Ner protein.

Binding of purified phage Mu Ner protein to a series of DNA fragments was investigated in order to determine the length requirements for tight specific binding. Gel retardation experiments with wild-type 307 base pair (bp) Mu DNA and shorter, synthetic oligonucleotides were performed, and apparent dissociation constants (KappD) were determined from the half-saturation point. While Ner formed four complexes with the 307 bp DNA fragment, only one complex was observed with the shorter DNAs. The 50 and 30 bp fragments had KappD values of 5 and 20 nM, respectively. Ner binding was progressively weaker with decreasing size of the DNA fragments, with no binding observed for 12mers. The shortest DNA fragments which bound well were two 18 bp fragments for which KappD values were in the range of 50-100 nM. The stoichiometry of Ner complexes with the 30 and 18 bp fragments was determined using a modified Ferguson method. Ner was found to form a tetramer on the 30 bp DNA and a dimer on the 18 bp DNA, which makes the latter a good candidate for the study of a Ner-DNA complex by NMR. In order to clarify which DNA regions were important for Ner-DNA binding, hydroxyl radical footprinting was performed for a range of Ner concentrations from 30 to 500 nM. The footprint revealed that Ner contacts the DNA backbone every 12-13 bp, on both strands of the DNA. The order in which protected regions appeared with increasing protein concentration indicated that two Ner monomers bound to DNA simultaneously. A model of Ner binding to DNA is proposed on the basis of these results.

Bacteriophage mu↗

PubMed1990

Phase transitions of concentrated DNA solutions in low concentrations of 1:1 supporting electrolyte.

Transitions between isotropic and liquid crystalline phases of concentrated solutions of DNA with an average contour length (500 A) near the persistence length were examined in 0.01 M supporting 1:1 electrolyte (predominantly NaCl). A quantitative phase diagram describing the transitions occurring over a DNA concentration range from 100 to 290 mg/mL and temperatures from 20 to 60 degrees C was constructed from solid-state 31P-nmr data and examination of the morphologies of the mesophases by polarized light microscopy. Three anisotropic phases were observed in solutions with DNA concentrations of 160-290 mg/mL: an unidentified, weakly birefringent phase termed "precholesteric," a true cholesteric phase with pitch approximately 2 microns, and a third, presumably more highly ordered phase. Comparison with previous studies showed that the critical concentration for anisotropic phase formation and the nature of the phases formed by these DNA molecules are not strongly affected by decreasing the supporting electrolyte concentration from approximately 0.2 M to 10 mM. There are, however, profound effects of decreasing the supporting electrolyte concentration on the width of the transition from isotropic to totally anisotropic solutions, and the nature of the transitions between phases. Decreasing the supporting electrolyte concentration significantly increases the concentration range of persistence of the isotrophic phase, and results in the formation of triphasic solutions (isotropic and two liquid crystalline phases). Values of the critical DNA concentrations for anisotropic phase formation from the theory of A. Stroobants et al. [(1986) Macromolecules 19, 2232 to 2238] were found to be significantly lower than the observed values for any reasonable estimate of the effective radius, probably because of the relatively short lengths of DNA fragments examined in the present study. Comparison of the experimentally determined DNA concentrations required for anisotropic phase formation with the values predicted from Flory's lattice statistics theory, which explicitly considers the rod length, permitted estimation of the effective DNA radius. The estimated radius was inconsistent with effective radii calculated from Poisson-Boltzmann (P-B) theory based on a supporting electrolyte concentration of 10 mM, but was in fair agreement with P-B theory assuming that Na+ DNA contributes approximately 0.24 Na+ counterions/nucleotide to the effective free sodium ion concentration.

DNA↗

PubMed1990

A 23Na-NMR study of sodium-DNA interactions in concentrated DNA solutions at low-supporting electrolyte concentration.

Aqueous solutions of DNA fragments with a contour length (500 A) near the persistence length at DNA concentrations ranging from 10 to 290 mg/mL solvent and a constant supporting electrolyte concentration of 0.01 M (predominantly NaCl) were examined by 23Na-nmr spectroscopy at temperatures of 20, 40, and 60 degrees C. Over the higher portion of this concentration range (greater than 100 mg/ml) the DNA solutions undergo a complex series of transitions between different anisotropic, liquid crystalline phases (T. E. Strzelecka and R. L. Rill, Biopolymers, in press). Counterions in solutions of strong polyelectrolytes are usually described in terms of a two-state model as free or "bound" (influenced by the electrostatic field of the polyanion). The longitudinal relaxation rate (R1 = 1/T1) at all DNA concentrations decreased with increasing temperature, demonstrating fast exchange between free and bound counterions. R1 increased nearly linearly with increasing DNA phosphate/sodium ratio in the isotropic domain until the onset of anisotropic phase formation, in agreement with similar nmr studies conducted at low DNA concentrations. The value of R1,b = 194 +/- 7 Hz obtained for the isotropic phase from 10 to 100 mg DNA/mL at 20 degrees C was in agreement with values reported previously. A nonlinear increase in R1 with DNA concentration was observed upon onset of anisotropic phase formation, indicating an increase in the product of the fraction of bond ions times their relaxation rate (r.R1,b). The spectral lineshape of all isotropic samples was Lorentzian. Spectra of anisotropic samples exhibited low magnitude quadrupole splitting of less than or equal to 400 Hz correlated with appearance of a cholesteric phase with pitch approximately 2 microns. The magnitude of the quadrupole splitting decreased with increasing DNA concentration at low temperatures and increased with concentration at high temperatures. At all concentrations the quadrupole splitting decreased then increased with temperature. These temperature- and concentration-dependent changes in quadrupole splitting are consistent with an angle between the DNA helix axis and the principal component (VZZ) of the local electric field gradient tensor near the "magic angle" of 54.7 degrees.

Animals↗

PubMed1988

Multiple liquid crystal phases of DNA at high concentrations.

DNA packaging in vivo is very tight, with volume concentrations approaching 70% w/v in sperm heads, virus capsids and bacterial nucleoids. The packaging mechanisms adopted may be related to the natural tendency of semi-rigid polymers to form liquid crystalline phases in concentrated solutions. We find that DNA forms at least three distinct liquid crystalline phases at concentrations comparable to those in vivo, with phase transitions occurring over relatively narrow ranges of DNA concentration. A weakly birefringent, dynamic, 'precholesteric' mesophase with microscopic textures intermediate between those of a nematic and a true cholesteric phase forms at the lowest concentrations required for phase separation. At slightly higher DNA concentrations, a second mesophase forms which is a strongly birefringent, well-ordered cholesteric phase with a concentration-dependent pitch varying from 2 to 10 micron. At the highest DNA concentrations, a phase forms which is two-dimensionally ordered and resembles smectic phases of thermotropic liquid crystals observed with small molecules.

Crystallization↗