Optically detected magnetic resonance of tryptophan residues in complexes formed between a bacterial single-stranded DNA binding protein and heavy atom modified poly(uridylic acid)

Optically detected magnetic resonance (ODMR) methods were employed to study three single-stranded DNA binding (SSB) proteins encoded by plasmids of enteric bacteria: pIP71a, R64, and F. Equilibrium binding isotherms obtained by fluorescence titrations reveal that the complexes of the plasmid SSB proteins with heavy atom modified polynucleotides are readily disrupted by salt. Since all the plasmid SSB proteins show limited solubility at low ionic strength (pIP71a greater than R64 greater than F), we were able to bind only the pIP71a protein to mercurated poly(uridylic acid) [poly(5-HgU)] and brominated poly(uridylic acid) [poly(5-BrU)]. ODMR results reveal the existence of at least one heavy atom perturbed, red-shifted, stacked Trp residue in these complexes. Amplitude-modulated phosphorescence microwave double resonance spectra display selectively the phosphorescence associated with Hg-perturbed Trp residue(s) in the pIP71a SSB protein-poly(5-HgU) complex, which has a broad, red-shifted 0,0-band. Our results suggest that Trp-135 in Escherichia coli SSB, which is absent in the plasmid-encoded SSB proteins, is located in a polar environment and is not involved in stacking interactions with the nucleotide bases. Phosphorescence spectra and lifetime measurements of the pIP71a SSB protein-poly (5-BrU) complex show that at least one Trp residue in the complex does not undergo stacking. This sets a higher limit of two stacking interactions of Trp residues with nucleotide bases in complexes of pIP71a SSB with single-stranded polynucleotides.     

Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides. A study by optical detection of magnetic resonance and site-selected mutagenesis

Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis. Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E. coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions. Wavelength-selected ODMR measurements on E. coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J. Biol. Chem. 262, 1725-1733). Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.     

Stacking interactions of tryptophan residues and nucleotide bases in complexes formed between Escherichia coli single-stranded DNA binding protein and heavy atom-modified poly(uridylic) acid. A study by optically detected magnetic resonance spectroscopy

The complexes formed between Escherichia coli single-stranded DNA binding protein (SSBP) and the heavy atom-modified single-stranded polynucleotides poly(5-BrU) and poly(5-HgU) are investigated using optically detected magnetic resonance (ODMR) methods. In these complexes the triplet state properties of the tryptophan residues are subjected to the external heavy atom effect generated by bromine and mercury atoms and are characterized by a shortened triplet state lifetime and the appearance of the otherwise dark [D] + [E] slow passage ODMR signal. These features provide direct evidence for close range interactions between tryptophan residue(s) and the nucleotide bases in the complexes. The extent of the triplet state lifetime reduction in the case of the SSBP-poly(5-HgU) complex together with steric considerations of the complex structure is consistent only with a van der Waals contact between the perturbed molecule and the heavy atom perturber by means of a stacking interaction. Fast passage ODMR measurements show a lifetime for a sublevel of the perturbed tryptophan chromophore(s) in this complex on the order of 1 ms. The amplitude-modulated phosphorescence microwave double resonance technique captures selectively the broadened and red-shifted phosphorescence spectrum of the heavy atom-perturbed tryptophan residue(s). This work supports a model for the binding of SSBP to single-stranded polynucleotides in which the bases are inserted into hydrophobic regions of the protein, where they are likely to undergo stacking interactions with the indole moiety of buried tryptophan residues.     

Energy transfer in complexes of E. coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid)

The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.     

Investigation of complexes formed between gene 32 protein from bacteriophage T4 and heavy-atom-modified single-stranded polynucleotides using optical detection of magnetic resonance

Optical detection of triplet-state magnetic resonance (ODMR) is employed to study the complexes formed between gene 32 protein (GP32), a single-stranded DNA-binding protein from bacteriophage T4, and the heavy-atom-derivatized polynucleotides poly(5-HgU) and poly(5-BrU). The triplet-state properties of some of the tryptophan (Trp) residues in the complexes are dramatically different from those in the free protein, in that they are subject to an external heavy-atom effect. Direct evidence for the presence of a heavy-atom effect, and hence a close-range interaction between mercurated or brominated nucleotide bases and Trp residues in the complex, is provided by the observation of the zero-field (D) + (E) ODMR transition of Trp, which is not normally observed in the absence of a heavy-atom perturbation. The amplitude-modulated phosphorescence-microwave double-resonance (AM-PMDR) technique is employed to selectively capture the phosphorescence spectrum originating from the heavy-atom-perturbed Trp residue(s) in the GP32-poly(5-HgU) complex. Arguments based on our experimental results lead to the conclusion that the heavy-atom perturbation arises from aromatic stacking interactions between Trp and mercurated bases. Wavelength-selected ODMR measurements reveal the existence of two environmentally distinct and spectrally different types of Trp in GP32. One of these types is perturbed selectively by the heavy atom and hence undergoes stacking interactions with the heavy-atom-derivatized bases of the polynucleotide while the second type of Trp residue is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

p10, a low molecular weight single-stranded nucleic acid binding protein of murine leukemia retroviruses, shows stacking interactions of its single tryptophan residue with nucleotide bases

Room temperature fluorescence and low-temperature phosphorescence studies of the association of p10, a basic low molecular weight single-stranded DNA binding protein isolated from murine leukemia viruses, point to the involvement of its single tryptophan residue in a close-range interaction with single-stranded polynucleotides. Optically detected triplet-state magnetic resonance (ODMR) techniques applied to the complex of p10 protein with the heavy atom derivatized polynucleotide poly(5-HgU) demonstrate the occurrence of stacking interactions of Trp35 with nucleic acid bases, thus agreeing with earlier reports that this residue is involved in the binding process [Karpel, R. L., Henderson, L. E., & Oroszlan, S. (1987) J. Biol. Chem. 262, 4961-4967].     

Binding of recA protein to single- and double-stranded polynucleotides occurs without involvement of its aromatic residues in stacking interactions with nucleotide bases

Phosphorescence and optically detected triplet state magnetic resonance (ODMR) spectroscopy studies of recA protein and its complexes with poly(5-HgU) and poly(dA-5BrdU) show that the two tryptophan residues are not involved in stacking interactions with the nucleotide bases of either single- or double-stranded polynucleotides. Solvent conditions which induce preferential binding to single-stranded ligands result in a shortening of the tyrosine phosphorescence lifetime, which is further reduced upon binding to poly(5-HgU). This suggests a change in the global conformation or self-aggregation state of the protein. Binding to poly(dA-5BrdU) induces small changes in the tryptophan zero field splittings of recA, but significant changes on those of 5BrdU, which are consistent with recA binding to the minor groove of the polynucleotide.     

An IncY plasmid-encoded single-stranded DNA-binding protein from Escherichia coli shows the identical pattern of stacked tryptophan residues as the chromosomal ssb gene product

In an extension of earlier studies on the Escherichia coli plasmid-encoded single-stranded DNA-binding proteins pIP71a SSB, F SSB and R64 SSB [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) Biochemistry 26, 3347-3354; Casas-Finet, J. R., Khamis, M. I., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) J. Biol. Chem. 262, 8574-8593], we have investigated the binding of pIP231a SSB to natural and heavy-atom-derivatized single-stranded homopolynucleotides. Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated. Furthermore, its complex with mercurated poly(uridylic acid) [poly(Hg5U)] shows a greater resistance to disruption by salt than the other plasmid SSB complexes. Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time-resolved optically detected triplet-state magnetic resonance (ODMR) techniques could be applied to the complex. These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB. Comparison of wavelength-selected ODMR results with those obtained for the poly(Hg5U) complex of a point-mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J. B. & Chase, J. W. (1987) J. Biol. Chem. 262, 10938-10945] confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not. This is the same distribution of stacked Trp residues found in Eco SSB. These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.     

Triplet state properties of tryptophan residues in complexes of mutated Escherichia coli single-stranded DNA binding proteins with single-stranded polynucleotides.

Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed.     

Continuous association of Escherichia coli single-stranded DNA binding protein with stable complexes of recA protein and single-stranded DNA

The single-stranded DNA binding protein of Escherichia coli (SSB) stimulates recA protein promoted DNA strand exchange reactions by promoting and stabilizing the interaction between recA protein and single-stranded DNA (ssDNA). Utilizing the intrinsic tryptophan fluorescence of SSB, an ATP-dependent interaction has been detected between SSB and recA-ssDNA complexes. This interaction is continuous for periods exceeding 1 h under conditions that are optimal for DNA strand exchange. Our data suggest that this interaction does not involve significant displacement of recA protein in the complex by SSB when ATP is present. The properties of this interaction are consistent with the properties of SSB-stabilized recA-ssDNA complexes determined by other methods. The data are incompatible with models in which SSB is displaced after functioning transiently in the formation of recA-ssDNA complexes. A continuous association of SSB with recA-ssDNA complexes may therefore be an important feature of the mechanism by which SSB stimulates recA protein promoted reactions.     

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.     

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.     

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.     

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.     

Optically detected magnetic resonance study of the interaction of an arsenic(III) derivative of cacodylic acid with EcoRI methyl transferase

The interaction of the enzyme Escherichia coli RI methyl transferase (methylase) with an arsenic(III) derivative of cacodylic acid has been investigated by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy in zero applied magnetic field. The reactive derivative (CH3)2AsSR is formed by the reduction of cacodylate by a thiol. The As(III) derivative binds to the enzyme by mercaptide exchange with a cysteine (Cys) residue located close to a tryptophan (Trp) site. The arsenical binding selectively induces an external heavy-atom effect, perturbing the nearby Trp residue in the enzyme. Zero-field splittings (ZFS) and total decay rate constants of the individual triplet-state sublevels of the Trp residue in the presence and absence of perturbation by As(III) have been determined. The perturbed Trp shows a large reduction in the overall decay lifetime compared with unperturbed Trp residue, exhibiting a high selectively for the Tx sublevel. This selectivity suggests that the As atom lies in the xz plane of the principal magnetic axis system of Trp, but not directly along the z (out-of-plane) axis. The accessibility of this enzyme binding site to the arsenical is decreased upon forming a ternary complex of methylase with sinefungin and a DNA oligomer, d[GCGAA(BrU)(BrU)CGC], containing two 5-bromouracil (BrU) bases in place of thymine within the hexadeoxynucleotide recognition sequence. This result indicates that the arsenical binding site in methylase which produces the Trp heavy-atom effect is protected from this ligand by ternary complex formation or the enzyme undergoes a conformation change, removing the Cys from the Trp site. This protection is also observed in fluorescence quenching experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan 54 and phenylalanine 60 are involved synergistically in the binding of E. coli SSB protein to single-stranded polynucleotides

The binding of both wild-type and point-mutated E. coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60. This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine.     

Triplet state sublevel kinetics of tryptophan 54 in the complex of Escherichia coli single-stranded DNA binding protein with single-stranded poly(deoxythymidylic) acid.

The individual sublevel kinetics of the lowest triplet state of tryptophan 54 (Trp 54) which is highly perturbed in the complex of Escherichia coli single-stranded DNA binding protein (Eco SSB) with poly(deoxythymidylic) acid (poly[dT]) have been studied by optically detected magnetic resonance (ODMR) spectroscopy. The triplet sublevel decay constants of Trp 54, kx, ky, kz, are 0.99, 0.072, and 0.045 s-1, respectively, in the poly(dT) complex of a point-mutated Eco SSB in which Trp 88 is substituted by phenylalanine. Tx is the only radiative triplet sublevel. Negative polarity of the Tx----Tz and Tx----Ty phosphorescence-detected ODMR signals results from the steady state population pattern, nx greater than ny, nz, and implies that the relations, px greater than or equal to 14py, and px greater than or equal to 22pz exist for the relative populating rates. Spin-orbit coupling between radiative singlet states and the Tx sublevel of the lowest triplet state of Trp 54 is enhanced selectively upon complexing of Eco SSB with poly(dT).     

Modulation of the affinity of the single-stranded DNA-binding protein of Escherichia coli (E. coli SSB) to poly(dT) by site-directed mutagenesis

A vector for site-directed mutagenesis and overproduction of the Escherichia coli single-stranded-DNA-binding protein (E. coli SSB) was constructed. An E. coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells. The vector was used to mutate specifically the Phe60 residue of E. coli SSB. Phe60 had been proposed to be located near the single-stranded-DNA-binding site. Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy. The affinity of the mutant E. coli SSB proteins for single-stranded DNA decreased in the order Trp greater than Phe (wild-type) greater than Tyr greater than Leu greater than His greater than Val greater than Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.     

Interactions of aromatic residues of proteins with nucleic acids. Fluorescence studies of the binding of oligopeptides containing tryptophan and tyrosine residues to polynucleotides.

The binding of oligopeptides of general structure Lys-X-Lys (where X is an aromatic residue) to several polynucleotides has been studied by fluorescence spectroscopy. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups as shown by the ionic strength and pH dependence of binding. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the nucleic acid bases with the aromatic amino acid whose fluorescence is quenched. Fluorescence data have been quantitatively analyzed according to a model involving these two types of complexes. Association constants and the size of binding sites have been determined. Stacking interactions are favored in single-stranded polynucleotides as compared to double-stranded ones. A short oligopeptide such as Lys-X-Lys is thus able to distinguish between single-stranded and double-stranded nucleic acids. Fluorescence results are compared to those obtained by proton magnetic resonance and circular dichroism.