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L B Overman

Publications and source records attributed to L B Overman.

8 recordsLinked to original sources
PubMed1996

A highly salt-dependent enthalpy change for Escherichia coli SSB protein-nucleic acid binding due to ion-protein interactions.

We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-drive; however, the value of [symbol see text] log Kobs/ [symbol see text] log [NaCl] is highly temperature- dependent, varying from -9.3 +/- 0.3 at 10 degrees C to -5.1 +/- 0.4 at 37 degrees C. This indicates that delta Hobs for SSB-poly(U) binding is strongly dependent on [NaCl]; based on van't Hoff analyses, delta Hobs varies from -57 +/- 3 kcal/mol at 0.18 M NaCl to -34 +/- 3 kcal/mol at 042 M NaCl ([symbol see text] delta Hobs/[symbol see text] log [NaCl] = 60 +/- 5 kcal/mol). However, [symbol see text] delta Hobs/[symbol see text] log [NaF] is independent of temperature (25-37 degrees C), indicating that the effect of [NaCl] on delta Hobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured delta Hobs and its dependence on [NaCl] for SSB binding dT(pT)69 by ITC and find delta Hobs = -144 +/- 4 kcal/mol (0.175 M NaCl, pH 8.1, 25 degrees C) and [symbol see text] delta Hobs/ [symbol see text] log [NaCl] = 46 +/- 2 kcal/ mol (0.175-2.0 M NaCl). These large effects of [NaCl] on delta Hobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with delta H > > 0. Effects of salt concentration on delta Hobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ of K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on delta Hobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to delta Hobs, as well as delta Hobs degrees and delta S(obs) degrees, in order to understand the bases for stability and specificity of protein-nucleic acid interactions.

Bacterial Proteins↗
PubMed1994

Linkage of pH, anion and cation effects in protein-nucleic acid equilibria. Escherichia coli SSB protein-single stranded nucleic acid interactions.

We have examined the linkage between pH and monovalent salt concentration (NaCl and NaF) on the equilibrium binding of the Escherichia coli SSB protein to single stranded poly(U) in its (SSB)65 binding mode. In this mode, single-stranded nucleic acid interacts with all four subunits of the SSB tetramer covering approximately 65 nucleotides and nearest-neighbor cooperative interactions can form between DNA bound SSB tetramers, although protein clusters are limited to dimers of tetramers (octamers). The intrinsic association equilibrium constant, K(obs), and the "limited" cooperativity parameter, omega T/O, have been determined from titrations that monitor the quenching of the SSB tryptophan fluorescence upon binding poly(U). The cooperativity parameter, omega T/O, is independent of salt concentration and type and increases only slightly with increasing pH. However, K(obs) decreases with increasing salt concentration due to a net release of ions accompanying complex formation. This net ion release has contributions from cation release from the nucleic acid as well as differential binding of both cations and anions to the protein. The dependence of K(obs) on [NaF] is independent of pH with delta logK(obs)/delta log[NaF] = -4.5(+/- 0.5). However, there is a strong linkage between the effects of [NaCl] and pH, such that (delta logK(obs)/delta log[NaCl]) ranges from -12.0(+/- 0.8) at pH 5.5, to -6.0(+/- 0.5) at pH 9.0 (at 25 degrees C). Thus Cl- release increases with decreasing pH due to a linkage between chloride binding and protonation of the protein, whereas there is essentially no release of F-. The linkages of ion concentration and pH on K(obs) can be described in terms of: (1) cation release from the polynucleotide; (2) release of Cl- from sites on the SSB tetramer that require protonation to bind Cl-; (3) binding of cations to sites on the SSB tetramer which require deprotonation for cation binding, and (4) required binding of two-to-three protons by the SSB tetramer in order to form the SSB-poly(U) complex. Thus, the influence of salt concentration on protein-nucleic acid equilibria can be quite complex with contributions from differential ion binding to both the protein and the nucleic acid; however, these can be resolved by examining the linked effects of pH and salt concentration on these interactions.

Anions↗
PubMed1988

Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects.

We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T. M. & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W. & Lohman, T. M. (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. "Reverse" titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 degrees C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations. Under all conditions in which "reverse" titrations were performed, we observe three distinct binding modes with site sizes of 35 +/- 2, 56 +/- 3, and 65 +/- 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transitions from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 degrees C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20 degrees C, pH 8.1) and observe an increase in s20,w, which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition. The transition between the (SSB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked.(ABSTRACT TRUNCATED AT 400 WORDS)

Anions↗
PubMed1988

Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity.

The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ("tetramer/octamer" model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperativity parameter, omegoT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of omegaT/O = 410 +/- 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25 degrees C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that alpha log Kobsd/alpha log [NaCl] = -7.4 +/- 0.5 for poly(U), -6.1 +/- 0.6 for poly(dA), and -6.2 +/- 0.3 for poly(A) (25.0 degrees C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure alpha log Kobsd/alpha log [NaBr] = -5.7 +/- 0.7, with a lower value of omega T/O = 130 +/- 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0 degrees C, pH 8.1) is Kobsd(dT) greater than Kobsd(dC) much greater than Kobsd(ss M13 DNA) greater than Kobsd(I) greater than Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) much greater than Kobsd(C).(ABSTRACT TRUNCATED AT 400 WORDS)

Anions↗
PubMed1986

Salt-dependent changes in the DNA binding co-operativity of Escherichia coli single strand binding protein.

The co-operative nature of the binding of the Escherichia coli single strand binding protein (SSB) to single-stranded nucleic acids has been examined over a range of salt concentrations (NaCl and MgCl2) to determine if different degrees of binding co-operativity are associated with the two SSB binding modes that have been identified recently. Quantitative estimates of the binding properties, including the co-operativity parameter, omega, of SSB to single-stranded DNA and RNA homopolynucleotides have been obtained from equilibrium binding isotherms, at high salt (greater than or equal to 0.2 M-NaCl), by monitoring the fluorescence quenching of the SSB upon binding. Under these high salt conditions, where only the high site size SSB binding mode exists (65 +/- 5 nucleotides per tetramer), we find only moderate co-operativity for SSB binding to both DNA and RNA, (omega = 50 +/- 10), independent of the concentration of salt. This value for omega is much lower than most previous estimates. At lower concentrations of NaCl, where the low site size SSB binding mode (33 +/- 3 nucleotides/tetramer) exists, but where SSB affinity for single-stranded DNA is too high to estimate co-operativity from classical binding isotherms, we have used an agarose gel electrophoresis technique to qualitatively examine SSB co-operativity with single-stranded (ss) M13 phage DNA. The apparent binding co-operativity increases dramatically below 0.20 M-NaCl, as judged by the extremely non-random distribution of SSB among the ssM13 DNA population at low SSB to DNA ratios. However, the highly co-operative complexes are not at equilibrium at low SSB/DNA binding densities, but are formed only transiently when SSB and ssDNA are directly mixed at low concentrations of NaCl. The conversions of these metastable, highly co-operative SSB-ssDNA complexes to their equilibrium, low co-operativity form is very slow at low concentrations of NaCl. At equilibrium, the SSB-ssDNA complexes seem to possess the same low degree of co-operativity (omega = 50 +/- 10) under all conditions tested. However, the highly co-operative mode of SSB binding, although metastable, may be important during non-equilibrium processes such as DNA replication. The possible relation between the two SSB binding modes, which differ in site size by a factor of two, and the high and low co-operativity complexes, which we report here, is discussed.

Allosteric Regulation↗
PubMed1985

Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration.

The binding properties of the Escherichia coli encoded single strand binding protein (SSB) to a variety of synthetic homopolynucleotides, as well as to single stranded M13 DNA, have been examined as a function of the NaCl concentration (25.0 degrees C, pH 8.1). Quenching of the intrinsic tryptophan fluorescence of the SSB protein by the nucleic acid is used to monitor binding. We find that the site size (n) for binding of SSB to all single stranded nucleic acids is quite dependent on the NaCl concentration. For SSB-poly(dT), n = 33 +/- 3 nucleotides/tetramer below 10 mM NaCl and 65 +/- 5 nucleotides/tetramer above 0.20 M NaCl (up to 5 M). Between 10 mM and 0.2 M NaCl, the apparent site size increases continuously with [NaCl]. The extent of quenching of the bound SSB fluorescence by poly(dT) also displays two-state behavior, 51 +/- 3% quenching below 10 mM NaCl and 83 +/- 3% quenching at high [NaCl] (greater than 01.-0.2 M NaCl), which correlates with the observed changes in the occluded site size. On the basis of these observations as well as the data of Krauss et al. (Krauss, G., Sindermann, H., Schomburg, U., and Maass, G. (1981) Biochemistry 20, 5346-5352) and Chrysogelos and Griffith (Chrysogelos, S., and Griffith, J. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,5803-5807) we propose a model in which E. coli SSB binds to single stranded nucleic acids in two binding modes, a low salt mode (n = 33 +/- 3), referred to as (SSB)33, in which the nucleic acid interacts with only two protomers of the tetramer, and one at higher [NaCl], n = 65 +/- 5, (SSB)65, in which the nucleic acid interacts with all 4 protomers of the tetramer. At intermediate NaCl concentrations a mixture of these two binding modes exists which explains the variable site sizes and other apparent discrepancies previously reported for SSB binding. The transition between the two binding modes is reversible, although the kinetics are slow, and it is modulated by NaCl concentrations within the physiological range. We suggest that SSB may utilize both binding modes in its range of functions (replication, recombination, repair) and that in vivo changes in the ionic media may play a role in regulating some of these processes.

DNA, Bacterial↗