Salt effects on protein-DNA interactions. The lambda cI repressor and EcoRI endonuclease

In this paper, finite-difference solutions to the nonlinear Poisson-Boltzmann (NLPB) equation are used to calculate the salt dependent contribution to the electrostatic DNA binding free energy for both the lambda cI repressor and the EcoRI endonuclease. For the protein-DNA systems studied, the NLPB method describes nonspecific univalent salt dependent effects on the binding free energy which are in excellent agreement with experimental results. In these systems, the contribution of the ion atmosphere to the binding free energy substantially destabilizes the protein-DNA complexes. The magnitude of this effect involves a macromolecular structure dependent redistribution of both cations and anions around the protein and the DNA which is dominated by long range electrostatic interactions. We find that the free energy associated with global ion redistribution upon binding is more important than changes associated with local protein-DNA interactions (ion-pairs) in determining salt effects. The NLPB model reveals how long range salt effects can play a significant role in the relative stability of protein-DNA complexes with different structures.     

Kinetic and thermodynamic studies of purine repressor binding to corepressor and operator DNA

The kinetic and thermodynamic parameters for purine repressor (PurR)-operator and PurR-guanine binding were determined using fluorescence spectroscopy and nitrocellulose filter binding. Operator binding affinity was increased by the presence of guanine as demonstrated previously (Choi, K. Y., Lu, F., and Zalkin, H. (1994) J. Biol. Chem. 269, 24066-24072; Rolfes, R. J., and Zalkin, H. (1990) J. Bacteriol. 172, 5637-5642), and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by approximately 10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is approximately 3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences.     

A variant of lambda repressor with an altered pattern of cooperative binding to DNA sites

The bacteriophage lambda repressor binds cooperatively to pairs of adjacent sites in the lambda chromosome, one repressor dimer binding to each site. The repressor's amino domain (that which mediates DNA binding) is connected to its carboxyl domain (that which mediates dimerization and the interaction between dimers) by a protease-sensitive linker region. We have generated a variant lambda repressor that lacks this linker region. We show that dimers of the variant protein are deficient in cooperative binding to sites at certain, but not all, distances. The linker region thus extends the range over which carboxyl domains of DNA-bound dimers can interact. In particular, the linker is required for cooperative binding to a pair of sites as found in the lambda chromosome, and thus is essential for the repressor's physiological function.     

Attempts to purify a membrane attached chromoid of bacteriophage lambda

Using methods which proved successful for the isolation of E. coli chromosome in a folded form (the E. coli chromoid), we have attempted to purity the "native" form of bacteriophage gamma chromosome from gamma infected cells. Upon sedimentation of lysates we find that phage DNA separates into two fractions, one of which cosediments with the bacterial chromoid ; the other sediments nearer to the top of gradient. Both fractions probably contain membrane-bound phage DNA, and both support the in vivo synthesis of phage DNA. The heavier fraction contains more closed circular parental DNA molecules than the lighter fraction. Formation of the latter is blocked by certain phage mutations. Being relatively free of bacterial DNA, the lighter fraction is suitable for further analysis.     

Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli

Replacement of Escherichia coli's RecBCD function with phage lambda's Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by a recBC sbcBC or recD strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The deltarecBCD::Plac-red kan replacement allele can be P1 transduced to other E. coli strains, making the hyper-Rec phenotype easily transferable.     

Structure and stability of monomeric lambda repressor: NMR evidence for two-state folding

The absence of equilibrium intermediates in protein folding reactions (i.e., two-state folding) simplifies thermodynamic and kinetic analyses but is difficult to prove rigorously. We demonstrate a sensitive method for detecting partially folded species based on using proton chemical shifts as local probes of structure. The coincidence of denaturation curves for probes throughout the molecule is a particularly stringent test for two-state folding. In this study we investigate a new form of the N-terminal domain of bacteriophage lambda repressor consisting of residues 6-85 (lambda 6-85) using nuclear magnetic resonance (NMR) and circular dichroism (CD). This truncated version lacks the residues required for dimerization and is monomeric under the conditions used for NMR. Heteronuclear NMR was used to assign the 1H, 15N, and backbone 13C resonances. The secondary and tertiary structure of lambda 6-85 is very similar to that reported for the crystal structure of the DNA-bound 1-92 fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196], as judged by analysis of chemical shifts, amide hydrogen exchange, amide-alpha coupling constants, and nuclear Overhauser enhancements. Thermal and urea denaturation studies were conducted using the chemical shifts of the four aromatic side chains as local probes and the CD signal at 222 nm as a global probe. Plots of the fraction denatured versus denaturant concentration obtained from these studies are identical for all probes under all conditions studied. This observation provides strong evidence for two-state folding, indicating that there are no populated intermediates in the folding of lambda 6-85.     

Heat and cold denatured states of monomeric lambda repressor are thermodynamically and conformationally equivalent

Although the denaturation of proteins by low temperatures is a well-documented phenomenon, little is known about the molecular details of the process. In this study, the parameters describing the denaturation thermodynamics of residues 6-85 of the N-terminal domain of lambda repressor have been determined by fitting the three-dimensional thermal-urea denaturation surface obtained by circular dichroism. The shape of the surface shows cold denaturation at low temperatures and urea concentrations above 2 M, which allows accurate determination of the apparent heat capacity of denaturation (delta Cp). Denaturation curves based on aromatic 1H NMR spectra give identical denaturation curves, confirming purely twostate folding under all conditions studies. The denaturation surface can be fit with constant delta Cp and delta In KD/delta[urea] (KD is the equilibrium constant for denaturation), consistent with a thermodynamically invariant denatured state. In addition, the aromatic 1H NMR spectrum of the cold denatured state at 0 degree C in 3 M uea is essentially identical to the spectrum at 70 degree C in 3 M urea. These observations indicate that the structures of the cold and heat denatured states, in the presence of 3 M urea, are thermodynamically and conformationally equivalent.     

Structural characterization of synthetic model peptides of the DNA-binding cI434 repressor by electrospray ionization and fast atom bombardment mass spectrometry

The structural characterization of two synthetic model peptides of the cI434 repressor is described. Unequivocal determination of the structure was achieved by means of electrospray ionization mass spectrometry of the intact peptides and by fast atom bombardment mass spectrometric identification of complementary peptide fragments obtained by tryptic and chymotrypic digestion and partial separation by reversed-phase high-performance liquid chromatography. The results show the potential of this approach for characterizing synthetic peptides of relatively high molecular weight.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Inactivation of bacteriophage lambda, Escherichia coli, and Candida albicans by ozone

The effects of ozone (O3) on three types of microbes were studied. Test suspensions were exposed to 600 ppm O3 at room temperature. Control experiments were performed under identical conditions using oxygen gas. Bacteriophage lambda was completely inactivated at 10 min while Escherichia coli and Candida albicans were only inactivated by factors of 10(5) and 10(4) respectively at 40 min. Exposure of a mixed microbial suspension to O3 for 5 min resulted in 100% killing of bacteriophages while the viability of E. coli remained unchanged. Various body fluids containing phages were exposed to O3. Compared to buffered solution, the decrease in phage titers was significantly slower in whole blood, plasma, and albumin. Both E. coli and C. albicans had increased production of thiobarbituric-acid-reactive substances with increased O3 exposure. 3H-labelled amino acids were incorporated into E. coli. O3 treatment resulted in a loss of radioactivity, indicating leakage of cytoplasmic contents. The data indicate that microbes are inactivated by O3 at different rates, possibly related to differential membrane permeability. The milieu in which microbes are present determines the effectiveness and outcome of O3 treatment.