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W Bujalowski

Publications and source records attributed to W Bujalowski.

52 records · Page 3Linked to original sources
PubMed1989

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. I. Evidence and a quantitative model.

The interaction of the Escherichia coli single strand binding (SSB) protein with single-stranded DNA is complex, since a number of different binding modes have been observed, with different DNA site sizes and binding properties and the transitions among these binding modes are strongly influenced by solution conditions in vitro. Recent experiments have suggested the existence of negative co-operativity among the multiple DNA binding sites within individual SSB tetramers. In order to probe this negative co-operativity, we have examined the binding of a series of oligonucleotides of varying length, using the quenching of the intrinsic SSB protein fluorescence to monitor binding. The stoichiometries for saturation of the SSB tetramer are 4, 2, 2, 1 and 1, for the oligonucleotides, dT(pT)N-1, with N = 16, 28, 35, 56 and 70, respectively, indicating that one molecule of either dT(pT)27 or dT(pT)34 interacts with two SSB subunits, whereas one molecule of dT(pT)15 interacts with only a single subunit. Saturation of the SSB tetramer with dT(pT)15, dT(pT)34, dT(pT)69 or poly(dT) results in 85 to 90% quenching of the SSB fluorescence, whereas saturation with dT(pT)27 or dT(pT)55 results in only 80% and 72% quenching, respectively. Therefore, a single-stranded DNA of at least 64 nucleotides is required to wrap around an SSB tetramer fully and interact with all four subunits. A quenching of 50(+/- 2)% is observed upon filling only half of the subunits with either one molecule of dT(pT)34 or two molecules of dT(pT)15, which agrees with the quenching and site size observed in the (SSB)35 polynucleotide binding mode. Direct binding measurements indicate that the binding of dT(pT)27 to its second site is influenced by the oligonucleotide that occupies the first binding site (either dT(pT)27 or dT(pT)34), providing proof for the existence of a true negative co-operativity. This negative co-operativity is observed also for the binding of the shorter oligonucleotide, dT(pT)15. A statistical thermodynamic ("square") model gives an excellent description of the binding of all oligonucleotides possessing multiple sites on the SSB tetramer, based on only two interaction constants, the intrinsic binding constant, KN, and the negative co-operativity parameter, sigma N. These data indicate that the binding sites (subunits) on the unliganded SSB tetramer are all equivalent, but that a non-equivalence between dimers of subunits within the tetramer is induced upon binding ssDNA.

Bacterial Proteins↗
PubMed1989

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects.

We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant KN, and the negative co-operativity constant, sigma N. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. delta log KN/delta log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that delta Ho = -26(+/- 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. sigma N less than 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of sigma 16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for sigma 16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

Bacterial Proteins↗
PubMed1989

On the cooperative binding of large ligands to a one-dimensional homogeneous lattice: the generalized three-state lattice model.

We present the general secular equation for three-state lattice models for the cooperative binding of large ligands to a one-dimensional lattice. In addition, a closed-form expression for the isotherm is also obtained, that can be used with all values of the cooperativity parameter omega(0 less than omega less than infinity) thus eliminating the need for multiple equations.

Chemical Phenomena↗
PubMed1988

Negative cooperativity within individual tetramers of Escherichia coli single strand binding protein is responsible for the transition between the (SSB)35 and (SSB)56 DNA binding modes.

We have examined the binding of the oligonucleotide dT (pT)34 to the Escherichia coli SSB protein as a function of NaCl and MgCl2 concentration (25 degrees C, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence. We find two binding sites for dT(pT)34 per single strand binding (SSB) protein tetramer, with each site possessing widely different affinities depending on the salt concentration. At 200 mM NaCl, we observe nearly stoichiometric binding of dT(pT)34 to both binding sites within the SSB tetramer, although a difference in the affinities is still apparent. However, when the NaCl concentration is lowered, the overall affinity of dT(pT)34 for the second site on the SSB tetramer decreases dramatically. At 1.5 mM NaCl, only a single molecule of dT(pT)34 can bind per SSB tetramer, even with a 10-fold molar excess of dT(pT)34. MgCl2 is effective at 100-fold lower concentrations than NaCl in promoting the binding of the second molecule of dT(pT)34. This binding behavior reflects an intrinsic property of the SSb tetramer, since it is also observed upon binding of smaller oligonucleotides, and the simplest explanation is that a salt-dependent negative cooperativity exists between DNA binding sites within the SSB tetramer. This phenomenon is also responsible for the transition between the two SSB-single strand (ss) polynucleotide binding modes that cover 35 and 56 nucleotides per tetramer [Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802]. Extreme negative cooperativity stabilizes the (SSB)35 binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
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↗
PubMed1988

Contributions to selective binding of aromatic amino acid residues to tRNA(Phe).

The binding of aromatic amino acid amides to two fragments of tRNA(Phe) has been measured, in order to explore contributions to selective interactions of amino acid residues with tRNA(Phe). Measurements of the Wye base fluorescence demonstrate that the isolated anticodon loop of tRNA(Phe) (15 nucleotide residues) has a binding constant K = 75 M-1 for Phe-amide, which is only slightly lower than that observed previously for the complete tRNA(Phe) (K = 100 M-1). The hexamer GmAAYA psi - possessing most residues of the loop, but without loop structure - shows a further reduced binding constant of 42 M-1. According to these data, part of the selective interaction results from the special loop structure and another part probably from the nature of the Wye base. The particular influence of the loop structure is also demonstrated by binding experiments performed in the presence of Mg2+. As expected, addition of Mg2+ decreases the binding affinity of the aromatic amino acids amides. Moreover, Mg2+ at concentrations greater than or equal to 0.3 mM induces cooperative binding of the amides to the anticodon loop similar to that found previously for the complete tRNA(Phe), whereas the hexamer does not show any indication for cooperativity. A special coupling of Mg2+ and amide binding to the anticodon loop is also indicated by inhibition of Mg2+ binding in the presence of amides, which is much stronger than expected for a simple salt effect. These results demonstrate a complex coupling of Mg2+ and amide binding to different conformational states of the anticodon loop, which resembles an allosteric type of reaction mechanism.

Amides↗
PubMed1987

Limited co-operativity in protein-nucleic acid interactions. A thermodynamic model for the interactions of Escherichia coli single strand binding protein with single-stranded nucleic acids in the "beaded", (SSB)65 mode.

We present a statistical thermodynamic model ("tetramer/octamer" model) that describes the equilibrium binding of the Escherichia coli single strand binding (SSB) protein to single-stranded nucleic acids in its "beaded" binding mode, which seems to be equivalent to the high site size, (SSB)65 binding mode. The method of sequence-generating functions is used to derive the model, which accounts for the observation that clustering of bound SSB tetramers is limited to the formation of octamers, which have been observed as "beads" in the electron microscope. The model also accounts for the overlap of potential protein binding sites on the nucleic acid. The "tetramer/octamer" model is fully described by only three parameters: the site size, n; the intrinsic equilibrium constant, K; and the co-operativity parameter, omega, and we obtain exact, closed form expressions for the binding isotherm as well as the distribution of DNA-bound SSB tetramers and octamers. The closed form expressions allow one to calculate easily average binding properties and analyze experimental binding isotherms to obtain estimates of K and omega. In order to test the tetramer/octamer model, we have determined the equilibrium binding isotherm for the E. coli SSB protein-poly(U) interaction in 0.2 M-NaCl over a wide range of binding densities. These are conditions in which the low co-operativity (SSB)65 binding mode solely exists. The tetramer/octamer model provides a much better description of the experimental isotherm over the entire binding density range than a model that assumes the formation of clusters of unlimited size. A co-operativity parameter of omega = 420 +/- 80 provides a good fit to data for SSB binding to poly(dA) and poly(U), corresponding to an interaction free energy of -3.6 kcal/mol of SSB octamer formed. On the basis of this moderate value of omega, the tetramer/octamer model predicts that at low to intermediate binding densities, a significant fraction of bound SSB exists in the form of tetramers co-existing with octamers. In the case of E. coli SSB protein binding in the "beaded", (SSB)65 mode this model provides a significant improvement over previous treatments which assume unlimited nearest-neighbor interactions, since the binding parameters, K and omega, represent physically meaningful interaction constants rather than fitting parameters.

Allosteric Regulation↗
PubMed1987

A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions.

We describe a general method for the analysis of ligand-macromolecule binding equilibria for cases in which the interaction is monitored by a change in a signal originating from the ligand. This method allows the absolute determination of the average degree of ligand binding per macromolecule without any assumptions concerning the number of modes or states for ligand binding or the relationship between the fractional signal change and the fraction of bound ligand. Although this method is generally applicable to any type of signal, we discuss the details of the method as it applies to the analysis of binding data monitored by a change in fluorescence of a ligand upon binding to a nucleic acid. We apply the analysis to the equilibrium binding of Escherichia coli single-strand binding (SSB) protein to single-stranded nucleic acids, which is monitored by the quenching of the intrinsic tryptophan fluorescence of the SSB protein. With this method, one can quantitatively determine the relationship between the fractional signal change of the ligand and the fraction of bound ligand, LB/LT, and rigorously test whether the signal change is directly proportional to LB/LT. For E. coli SSB protein binding to single-stranded nucleic acids in its (SSB)65 binding mode [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Chrysogelos, S., & Griffith, J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5803], we show that the fractional quenching of the SSB fluorescence is equal to the fraction of bound SSB.

Bacterial Proteins↗
PubMed1986

Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA.

Four distinct binding modes for the interaction of Escherichia coli single-strand binding (SSB) protein with single-stranded (ss) DNA have been identified on the basis of quantitative titrations that monitor the quenching of the SSB protein fluorescence upon binding to the homopolynucleotide poly(dT) over a range of MgCl2 and NaCl concentrations at 25 and 37 degrees C. This is the first observation of multiple binding modes for a single protein binding to DNA. These results extend previous studies performed in NaCl (25 degrees C, pH 8.1), in which two distinct SSB-ss DNA binding modes possessing site sizes of 33 and 65 nucleotides per bound SSB tetramer were observed [Lohman, T.M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603]. Each of these binding modes differs in the number of nucleotides occluded upon interaction with ss DNA (i.e., site size). Along with the previously observed modes with site sizes of 35 +/- 2 and 65 +/- 3 nucleotides per tetramer, a third distinct binding mode, at 25 degrees C, has been identified, possessing a site size of 56 +/- 3 nucleotides per bound SSB tetramer, which is stable over a wide range of MgCl2 concentrations. At 37 degrees C, a fourth binding mode is observed, possessing a site size of 40 +/- 2 nucleotides per tetramer, although this mode is observable only over a small range of salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Single-Stranded↗
PubMed1986

Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding.

The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticodon↗
PubMed1986

Codon-induced association of the isolated anticodon loop of tRNAPhe.

The binding of the codon UUC to the isolated anticodon loop of tRNAPhe (yeast) has been studied as a model of codon recognition by a simple adaptor. Fluorescence titrations demonstrate that UUC binds to the isolated anticodon loop with an equilibrium constant of 1.4 X 10(3) M-1 (at 7.2 degrees C). Equilibrium sedimentation curves reveal that UUC binding induces association of anticodon loops beyond the dimer stage. A set of complete sedimentation curves obtained for various reactant concentrations was analyzed according to a model with an infinite number of subsequent association steps for UUC-anticodon loop complexes and with equal affinity for each step. The coupling of association and sedimentation was considered quantitatively, and the information resulting from conservation of mass was used by integration. According to this procedure, the experimental data can be described by an isodesmic association constant of 8 X 10(3) M-1 with satisfactory accuracy. Temperature-jump relaxation detected by fluorescence measurements provides independent evidence for codon-induced association of the anticodon loop. The data are consistent with the following mechanism: UUC preferentially binds to one of two loop conformations with a rate constant of 4.5 X 10(6) M-1 s-1; the UUC-anticodon loop complex undergoes association with a rate constant of 6.5 X 10(6) M-1 s-1. The reactions observed for the isolated anticodon loop are surprisingly similar to those observed previously for the complete tRNA, suggesting that simple hairpin loops are appropriate adaptors for a translation process at an early stage of evolution; the codon-induced association of the hairpin loop should be very useful to facilitate the transfer of cognate amino acids during translation.

Anticodon↗
PubMed1984

Selective binding of amino acid residues to tRNAPhe.

The interaction of amino acid amides with tRNAPhe was studied by measurements of the Wye base fluorescence. Binding of phenylalanine-, tyrosine- and tryptophan-amides leads to considerable quenching, whereas the amides of e.g. glycine and leucine do not induce quenching under the same conditions. Binding constants at 0.13 M salt - 100 M-1 for Phe-, 110 M-1 for Tyr- and 300 M-1 for Trp-amide - are about a factor of 6 higher than those evaluated from independent measurements for binding to simple single-stranded polynucleotides; the corresponding factor is 10 for double-stranded polynucleotides. Since the apparent enthalpy changes derived from measurements at different temperatures remains relatively low (-9 to -20 kJ/mol), the increased affinity appears to be mainly due to an increase of the entropy changes. Titration experiments performed in the presence of Mg2+ indicate cooperative interactions of the aromatic residues with the anticodon loop that are consistent with preferential binding to one of two loop conformations. Measurements of binding constants at different pH-values indicate the protonation of a tRNA residue in the tryptophanamide-tRNAPhe complex characterised by a pK value of about 7.0.

Allosteric Regulation↗
PubMedDate not supplied

Selective binding of amino acid residues to tRNA molecules detected by anticodon-anticodon interactions.

Anticodon-anticodon pairing of complementary tRNA's has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA. This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons. Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNAPhe.tRNAGlu pairing constant Kp, whereas Kp remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide. The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNAPhe. The binding constants evaluated according to this model (Phe-amide 120 M-1, Tyr-amide 160 M-1 and Trp-amide 580 M-1) are consistent with values obtained independently by fluorescence titrations with tRNAPhe. Selective binding of these amino acid residues to tRNAPhe is deduced from the observed concentration dependence which is not compatible with a corresponding binding process to tRNAGlu. Addition of glutamic acid diamide induces an increase of the tRNAPhe.tRNAGlu pairing constant, which is however equivalent to that observed for tRNAPhe.tRNALys pairing and thus does not demonstrate a selective binding to tRNAGlu. The pairing of tRNAPhe with tRNAGlu is strongly enhanced by addition of Mg2+ or spermine. Evaluation of the Mg2+ data by a site model leads to constants of 360 M-1 for the binding of Mg2+ to monomer tRNA and 3000 M-1 for the binding of Mg2+ to the tRNAPhe.tRNAGlu dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗