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.     

Microsecond dynamics of protein-DNA interactions: direct observation of the wrapping/unwrapping kinetics of single-stranded DNA around the E. coli SSB tetramer

The Escherichia coli single-stranded DNA binding protein (SSB) binds selectively to single-stranded (ss) DNA intermediates during DNA replication, recombination and repair. Each subunit of the homo-tetrameric protein contains a potential ssDNA binding site, thus the protein can bind to ssDNA in multiple binding modes, one of which is the (SSB)(65) mode, in which a 65 nucleotide stretch of ssDNA interacts with and wraps around all four subunits of the tetramer. Previous stopped-flow kinetic studies of (SSB)(65) complex formation using the oligodeoxynucleotide, (dT)70, were unable to resolve the initial binding step from the rapid wrapping of ssDNA around the tetramer. Here we report a laser temperature-jump study with resolution in the approximately 500 ns to 4 ms time range, which directly detects these ssDNA wrapping/unwrapping steps. Biphasic time courses are observed with a fast phase that is concentration-independent and which occurs on a time-scale of tens of microseconds, reflecting the wrapping/unwrapping of ssDNA around the SSB tetramer. Analysis of the slower binding phase, in combination with equilibrium binding and stopped-flow kinetic studies, also provides evidence for a previously undetected intermediate along the pathway to forming the (SSB)(65) complex.     

Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT)15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT)15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT)15 binding to the tetramer in each phase. However, the intrinsic binding constant, K16, for the SSB-1 protomer-dT(pT)15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, sigma 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT)15 without negative co-operativity; however, the intrinsic affinity of dT(pT)15 for the monomer is a factor of approximately 10 lower than for the protomer (50 mM-NaCl, pH 8.1, 25 degrees C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.     

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.     

Plasmodium falciparum SSB tetramer binds single-stranded DNA only in a fully wrapped mode

The tetrameric Escherichia coli single-stranded DNA (ssDNA) binding protein (Ec-SSB) functions in DNA metabolism by binding to ssDNA and interacting directly with numerous DNA repair and replication proteins. Ec-SSB tetramers can bind ssDNA in multiple DNA binding modes that differ in the extent of ssDNA wrapping. Here, we show that the structurally similar SSB protein from the malarial parasite Plasmodium falciparum (Pf-SSB) also binds tightly to ssDNA but does not display the same number of ssDNA binding modes as Ec-SSB, binding ssDNA exclusively in fully wrapped complexes with site sizes of 52-65 nt/tetramer. Pf-SSB does not transition to the more cooperative (SSB)(35) DNA binding mode observed for Ec-SSB. Consistent with this, Pf-SSB tetramers also do not display the dramatic intra-tetramer negative cooperativity for binding of a second (dT)(35) molecule that is evident in Ec-SSB. These findings highlight variations in the DNA binding properties of these two highly conserved homotetrameric SSB proteins, and these differences might be tailored to suit their specific functions in the cell.     

Association-dissociation of mammalian brain glutamine synthetase: Effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn₄E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982)Biochemistry24, 6389–6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 μg/ml (20 nm octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn₄EMn₄ could be activated further by Mn(II) to form a species Mn₄EMn₄Mn₈ that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn₄EMn₄, associated to octamers more extensively than Mn₄E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and l-glutamine, or ATP and l-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a K_d value of ca. 2.5 × 10^?6m, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 ± 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 m urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (±20) for tetramers in solution. Light-scattering experiments showed that, with 1.7–2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn₄EMn₄Mn₈)_n, where $\text{n}\text{?}\text{? 5}$. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer. Analysis of various stereochemical models for the interaction of subunit domains (assuming identical subunits) within tetramers, between tetramers in the octamers, and between octamers indicate that the data are most consistent with isologous, rather than heterologous, interactions to produce octamer. These analyses also predict that formation of oligomers from cubic octamers through weaker, Mn(II)-dependent interactions also are most likely to occur via isologous domains. The available electron micrographic evidence support these hypothetical models. Interactions within tetramers are stronger than those between tetramers, which are stronger than those between octamers.     

Crystal structure of the lambda repressor C-terminal domain octamer.

The three-dimensional structure of the lambda repressor C-terminal domain (CTD) has been determined at atomic resolution. In the crystal, the CTD forms a 2-fold symmetric tetramer that mediates cooperative binding of two repressor dimers to pairs of operator sites. Based upon this structure, a model was proposed for the structure of an octameric repressor that forms both in the presence and absence of DNA. Here, we have determined the structure of the lambda repressor CTD in three new crystal forms, under a wide variety of conditions. All crystals have essentially the same tetramer, confirming the results of the earlier study. One crystal form has two tetramers bound to form an octamer, which has the same overall architecture as the previously proposed model. An unexpected feature of the octamer in the crystal structure is a unique interaction at the tetramer-tetramer interface, formed by residues Gln209, Tyr210 and Pro211, which contact symmetry-equivalent residues from other subunits of the octamer. Interestingly, these residues are also located at the dimer-dimer interface, where the specific interactions are different. The structures thus indicate specific amino acid residues that, at least in principle, when altered could result in repressors that form tetramers but not octamers.     

Plasmodium falciparum SSB tetramer wraps single-stranded DNA with similar topology but opposite polarity to E. coli SSB

Single-stranded DNA binding (SSB) proteins play central roles in genome maintenance in all organisms. Plasmodium falciparum, the causative agent of malaria, encodes an SSB protein that localizes to the apicoplast and likely functions in the replication and maintenance of its genome. P. falciparum SSB (Pf-SSB) shares a high degree of sequence homology with bacterial SSB proteins but differs in the composition of its C-terminus, which interacts with more than a dozen other proteins in Escherichia coli SSB (Ec-SSB). Using sedimentation methods, we show that Pf-SSB forms a stable homo-tetramer alone and when bound to single-stranded DNA (ssDNA). We also present a crystal structure at 2.1 ? resolution of the Pf-SSB tetramer bound to two (dT)(35) molecules. The Pf-SSB tetramer is structurally similar to the Ec-SSB tetramer, and ssDNA wraps completely around the tetramer with a "baseball seam" topology that is similar to Ec-SSB in its "65 binding mode". However, the polarity of the ssDNA wrapping around Pf-SSB is opposite to that observed for Ec-SSB. The interactions between the bases in the DNA and the amino acid side chains also differ from those observed in the Ec-SSB-DNA structure, suggesting that other differences may exist in the DNA binding properties of these structurally similar proteins.     

Association-dissociation of mammalian brain glutamine synthetase: effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn4E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982) Biochemistry 24, 6389-6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 micrograms/ml (20 nM octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn4EMn4 could be activated further by Mn(II) to form a species Mn4EMn4Mn8 that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn4EMn4, associated to octamers more extensively than Mn4E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and L-glutamine, or ATP and L-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a Kd value of ca. 2.5 X 10(-6) M, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 +/- 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 M urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (+/- 20) for tetramers in solution. Light-scattering experiments showed that, with 1.7-2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn4EMn4Mn8)n, where n greater than or equal to 5. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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)     

Co-axial association of recombinant eye lens aquaporin-0 observed in loosely packed 3D crystals

Aquaporin-0 (AQP0) is the major membrane protein in vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow inter-cellular spacing. We have obtained 3D crystals of recombinant bovine AQP0 that diffract to 7.0 A resolution. The crystal packing was determined by molecular replacement and shows that, within the cubic lattice, AQP0 tetramers are associated head-to-head along their 4-fold axes. Oligomeric states larger than the tetramer were also observed in solution by native gel electrophoresis and analytical ultracentrifugation methods. In the crystals, there are no direct contacts between octamers, and it can thus be inferred that crystalline order is mediated solely by the detergent belts surrounding the membrane protein. Across the tetramer-tetramer interface, extracellular loops A and C interdigitate at the center and the perimeter of the octamer, respectively. The octamer structure is compared with that of the recently determined structure of truncated ovine AQP0 derived from electron diffraction of 2D crystals. Intriguingly, also in these crystals, octamers are observed, but with significantly different relative tetramer-tetramer orientations. The interactions observed in the loosely packed 3D crystals reported here may in fact represent an in vivo association mode between AQP0 tetramers from juxtaposed membranes in the eye lens.     

Effect of Mg2+ on the DNA binding modes of the Streptococcus pneumoniae SsbA and SsbB proteins

The effect of Mg2+ on the binding of the Streptococcus pneumoniae single-stranded DNA binding (SSB) proteins, SsbA and SsbB, to various dT(n) oligomers was examined by polyacrylamide gel electrophoresis. The results were then compared with those that were obtained with the well characterized SSB protein from Escherichia coli, SsbEc. In the absence of Mg2+, the results indicated that the SsbEc protein was able to bind to the dT(n) oligomers in the SSB(35) mode, with only two of the four subunits of the tetramer interacting with the dT(n) oligomers. In the presence of Mg2+, however, the results indicated that the SsbEc protein was bound to the dT(n) oligomers in the SSB(65) mode, with all four subunits of the tetramer interacting with the dT(n) oligomers. The SsbA protein behaved similarly to the SsbEc protein under all conditions, indicating that it undergoes Mg2+ -dependent changes in its DNA binding modes that are analogous to those of the SsbEc protein. The SsbB protein, in contrast, appeared to bind to the dT(n) oligomers in an SSB(65)-like mode in either the presence or the absence of Mg2+, suggesting that it may not exhibit the pronounced negative intrasubunit cooperativity in the absence of Mg2+ that is required for the formation of the SSB(35) mode. Additional experiments with a chimeric SsbA/B protein indicated that the structural determinants that govern the transitions between the different DNA binding modes may be contained within the N-terminal domains of the SSB proteins.     

Histone interactions in solution and susceptibility to denaturation.

Histone interactions in solution may depend upon treatments used for purification. Optical rotatory dispersion and sedimentation-velocity measurements have been made in a reference solvent, before and after exposure to various treatments, to investigate histone susceptibility to irreversible denaturation. Some acid conditions and urea and guanidine solutions may denature. Interaction studies performed on nondenatured histones indicate that the dimer, (H4)(H3), and tetramer, (H4)2(H3)2, dissociate to monomers at low ionic strength. Sedimentation-velocity experiments suggest a model for the (H4)2(H3)2 tetramer, with a compact semispherical center and four protruding amino-terminal regions. Fractions H2a and H2b interact to form the mixed dimer in equilibrium with monomers. Fraction H2a self-associates readily to dimers, tetramers, and octamers, while fraction H1 associates only weakly to form dimers.     

Probing E. coli SSB protein-DNA topology by reversing DNA backbone polarity

Escherichia coli single-strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in two major modes, differing in occluded site size and cooperativity. The (SSB)₃₅ mode in which ssDNA wraps, on average, around two subunits is favored at low [NaCl] and high SSB/DNA ratios and displays high unlimited, nearest-neighbor cooperativity forming long protein clusters. The (SSB)₆₅ mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (>200 mM) and displays limited low cooperativity. Crystal structures of E. coli SSB and Plasmodium falciparum SSB show ssDNA bound to the SSB subunits (OB folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)₇₀ constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse-polarity (RP) phosphodiester linkage, either 3′-3′ or 5′-5′. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)₇₀ with a 3′-3′ polarity switch shows decreased affinity for EcSSB in the (SSB)₆₅ mode and lower cooperativity in the (SSB)₃₅ mode. However, (dT)₇₀ in which every phosphodiester linkage is reversed does not form a completely wrapped (SSB)₆₅ mode but, rather, binds EcSSB in the (SSB)₃₅ mode with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)₆₅ mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB folds if there is a change in ssDNA wrapping topology of the RP ssDNA.     

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.     

Structure and dynamics of the complex of single stranded DNA binding protein of Escherichia coli with circular single stranded DNA of filamentous phages

We have analyzed the equilibrium and nonequilibrium properties of the complex of the single stranded DNA binding protein of Escherichia coli (EcoSSB) and circular single stranded DNA of filamentous phages M13mp8 and F1 using static and dynamic light scattering, analytical ultracentrifugation and electron microscopy. Upon binding to the single stranded DNA the EcoSSB tetramer replaces an equivalent volume of water trapped within the coiled single stranded DNA and hinders the folding of the single stranded DNA into secondary structures at all salt concentrations. The salt dependent compaction of the stoichiometric complex can be described assuming a flexible polyelectrolyte chain. The solution structure of the macromolecular complex is a random coil and in the electron microscope a beaded flexible structure of the complex with a bead diameter of 6 nm appears at all salt concentrations used. The internal motions of the stoichiometric complex can be described by the Rouse-Zimm model of polymer dynamics. The segmental mobility of the complex can be correlated with changes in the binding site size of the EcoSSB tetramer; it indicates the presence of interactions between EcoSSB tetramers bound to single stranded DNA.     

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)     

SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。