Structural Features of phi 29 Single-stranded DNA-binding Protein
I. ENVIRONMENT OF TYROSINES IN TERMS OF COMPLEX FORMATION WITH DNA*

(Received for publication, April 1, 1996, and in revised form, October 2, 1996)

María S. Soengas Dagger §, C. Reyes Mateo , Margarita Salas Dagger par , A. Ulises Acuña and Crisanto Gutiérrez Dagger

From the Dagger  Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain and the  Instituto de Química Física Rocasolano (CSIC), Serrano 119, 28006 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage phi 29 is absolutely required for viral DNA replication in vivo. About ~95% of the intrinsic tyrosine fluorescence of phi 29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the phi 29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. phi 29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the phi 29 SSB fluorescence emission (Phi F = 0.067 ± 0.010), (ii) the fast motions of the tyrosine side chains (phi short = 0.14 ± 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that phi 29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and omega  (cooperativity) defining phi 29 SSB-poly(dT) interaction, the effective DNA binding constant, Keff = Komega , being largely independent of temperature. Altogether, the fluorescent properties of phi 29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.


INTRODUCTION

Interactions of nucleic acids and proteins provide the structural framework for a wide variety of biological processes. Among them, complexes formed by the association of single-stranded DNA-binding proteins (SSBs)1 with single-stranded DNA (ssDNA) are of particular interest. First, they modulate key processes of DNA metabolism: DNA replication and DNA repair (1, 2), transcription and translation regulation (3, 4), and recombination (5, 6, 7). Second, although all SSBs bind ssDNA with high affinity, the functional outcome of this interaction could be radically different. Thus, while most of them activate DNA replication, e.g. Escherichia coli SSB, T4 gp32, T7 gene 2.5 protein, phi 29 SSB, RP-A (reviewed in Ref. 1), some SSBs are inhibitory. Thus, SSBs of filamentous phages M13, fd and f1 or Pf3 block DNA replication by preventing the conversion of the viral strand into the replicative form (8). Third, detailed structural studies of a few SSBs have revealed that virtually each SSB interacts with DNA using a characteristic and largely specific set of contacts (9, 10, 11, 12, 13). As a consequence of this diversity, the molecular nature of the SSB-DNA interactions are less understood than those occurring with double-stranded DNA-binding proteins.

Bacillus subtilis phage phi 29 DNA replication requires a SSB. In vivo, the elongation process of DNA replication is dependent on the expression of a virally-encoded SSB protein, phi 29 SSB (reviewed in Refs. 14 and 15), the cellular SSB being apparently unable to support phi 29 DNA replication. In vitro, phi 29 SSB is essential to obtain full-length phi 29 DNA when the amount of DNA used as template is low (16), the closest condition to the initial steps of viral infection. Even when the amount of DNA template is high, phi 29 SSB is important for the in vitro system, increasing several times dNTP incorporation (17, 18). This stimulatory effect is related to binding to the ssDNA produced during phi 29 DNA replication (19), which, likely, protects it from nuclease degradation and prevents unproductive binding of the phi 29 DNA polymerase. phi 29 SSB also disrupts, by its helix-destabilizing activity, the secondary structure formed in ssDNA parts of phi 29 replicative intermediates. This activity has been proposed to account for the stimulation of the DNA elongation velocity by phi 29 DNA polymerase under conditions in which the DNA opening is impaired (20).

The thermodynamic parameters defining phi 29 SSB-ssDNA complex formation have been recently described: it binds to ssDNA with relatively low affinity (Keff = 105 M-1) and moderate cooperativity (omega  = 50-70), covering 3-4 nt per monomer, probably as a single array of protein units along the DNA (21). However, the precise mechanism that accounts for the requirement of phi 29 SSB during viral DNA replication is still unknown. The lack of significant sequence homology with other SSBs (18) has made difficult the identification of the phi 29 SSB domain(s) responsible for DNA binding and protein-protein contacts that lead to a stable protein-DNA interaction. Interestingly, complex formation occurs with significant alterations in the intrinsic tyrosine fluorescence of phi 29 SSB. Thus, 95% of the protein fluorescence is quenched upon binding (21), this value being one of the highest reported for a SSB, and similar to that observed for proteins that bind DNA through direct interactions with tyrosine residues (22). If the three tyrosines of phi 29 SSB (at positions 50, 57, and 76) were in fact forming part of the domain directly involved in ssDNA complex organization, they should not be highly restricted by interactions with other neighboring residues and, likely, placed in an accessible domain of the protein. To test this possibility, we have characterized the global spectroscopic properties of the tyrosines of phi 29 SSB by using complementary approaches: (i) steady-state and time-resolved measurements of the total emission and anisotropy of phi 29 SSB fluorescence, (ii) analysis of the resistance of phi 29 SSB tyrosines to undergo ionization by pH increase, and (iii) selective attenuation of the tyrosine fluorescence by small diffusible quenchers. In addition, the fluorescent properties of phi 29 SSB were also considered to study ssDNA binding under different temperatures. Our results regarding the mobility and accessibility of phi 29 SSB tyrosines strongly suggest a direct role of these residues in complex formation. In the accompanying paper (23), some of the fluorescence features of phi 29 SSB tyrosines are combined with other spectroscopic and hydrodynamic studies, to model the conformational properties of phi 29 SSB and the effect of complex formation on both phi 29 SSB and ssDNA structure.


MATERIALS AND METHODS

Buffers, Reagents, and Proteins

All the solutions were made with distilled, deionized (MilliQ) and sterilized water, and finally filtered through 0.45-µm filters using a Millex-HA Filter Unit (Millipore). The buffer used in time-resolved fluorescence analysis was 5 mM Tris-HCl, pH 7.5 (20 °C) (buffer A), while the buffer used in absorption and steady-state fluorescence studies was 50 mM Tris-HCl, pH 7.5 (20 °C), 4% glycerol (buffer B). Poly(dT) was from Pharmacia Biotech Inc. phi 29 SSB was purified by differential ammonium sulfate fractionation of phi 29-infected cellular extracts followed by phosphocellulose and DEAE-cellulose chromatography, essentially as described (20). Protein concentration was estimated by the Lowry (24) method, using bovine serum albumin as a standard.

Optical Absorption Measurements

Absorption spectra were carried out in a CARY 219 (Varian) at a constant temperature of 22 °C with cells of path-lengths from 2 to 10 mm.

Steady-state Fluorescence Spectroscopy

Measurements of the intrinsic tyrosine fluorescence of phi 29 SSB were recorded, unless otherwise stated, at 22 °C. Fluorescence excitation and emission spectra, solute quenching experiments, as well as ssDNA binding assays were performed in a Schoeffel Instrument Corp. RRS100 spectrofluorimeter (slits set to 2 nm), with a 2-mm path-length cell, and corrected for residual emission of the buffers. Fluorescence quantum yield and fluorescence emission anisotropy measurements were carried out in a SLM-8000D photon counting fluorimeter fitted with Glan-Thompson polarizers. The excitation and emission slits were 4 and 8 nm, respectively. When necessary, corrections for wavelength dependence of the instrument response were taken into account as described elsewhere (25).

The steady-state average fluorescence quantum yield (Phi FST) of the protein was calculated as in Demas and Crosby (26) using an aqueous solution of 5-methoxy-indole (5-MeOI) as reference (Phi REF = 0.28 ± 0.01; Ref. 27). Magic angle orientation of the polarizers was selected to avoid anisotropic effects (28).

The accessibility to solvent and the ionic environment of the fluorescent tyrosines of phi 29 SSB were analyzed by monitoring the fluorescence quenching (lambda exc = 276 nm, lambda em = 308 nm) induced by adding increasing amounts of stock solutions (2.5 M) of neutral (acrylamide) or charged quenchers, I- (as KI) and Cs+ (as CsCl) to the protein sample (7-11 µM). To prevent iodide oxidation, and avoid light dispersion and chemical reactions (29), Na2S2O3, at a final concentration of 200 µM, was added to the KI solution. Corrections for dilution and inner filter effect due to quencher absorption were made as described previously (21). Data were analyzed by Stern-Volmer plots of F0/F versus [Q], where F0 and F stand for the fluorescence intensities in the absence and in the presence of quencher, respectively, and [Q] is the quencher concentration. Since these plots were linear, the "static" quenching contribution was considered <10% (see "Discussion"), and the Stern-Volmer constant, Ksv, could be directly obtained from the slope of the line (see Refs. 30 and 31). This parameter represents the weighted sum of the individual constants of the emitting tyrosine residues and, therefore, is referred as Ksv,eff in the text. Ksv,eff was used to calculate the bimolecular rate constant of the quenching process, kq,eff, from the expression Ksv,eff = kq,eff·<A><AC>&tgr;</AC><AC>¯</AC></A>, where <A><AC>&tgr;</AC><AC>¯</AC></A> is the weighted average lifetime of phi 29 SSB fluorescence (see below). The fractional accessibility of the tyrosine residues to quencher was also estimated from the y axis intercept of double reciprocal plots as proposed by Lehrer (Ref. 29; see also Refs. 30, 31, 32). The solid angle Omega , under which the quencher can freely approach the tyrosine residues, was calculated as described (33), taking the values of the diffusion constants of the acrylamide, the iodide ion and the tyrosyl residues as 2 × 10-5 cm2 s-1, 1.5 × 10-5 cm2 s-1, and 0, respectively (33, 34). N-Acetyl-L-tyrosine amide (NAcTyrA) was taken as a control for a fully accessible residue.

The effect of temperature on complex formation with ssDNA was analyzed by monitoring the fluorescence intensity changes during direct titrations of a fixed amount of poly(dT), 30-40 µM (in nt), with increasing amounts of phi 29 SSB as described previously (Ref. 21 and references therein). The reaction buffer, buffer B, was adjusted to pH 7.5 at 20 °C. Since in the temperature range analyzed (10-42 °C) the pH variation (from pH 7.0 to 7.6) does not alter the global structural properties of phi 29 SSB (see accompanying paper (23)), no pH readjustments were made during the binding measurements. The thermodynamic parameters, nucleotide site size, n, intrinsic affinity constant, K, and cooperativity, omega , were obtained by fitting the experimental data to the theoretical ones given by the McGhee and von Hippel's (35) equations for binding of proteins to large lattices with overlapping binding sites (see Ref. 21, for further details). The enthalpy and the entropy of the binding process were approximated from the slope and the y axis intercept of the best fit of van't Hoff plots of lnKomega versus 1/T, as -Delta Ho/R and Delta So/R (R being the gas constant), respectively, since the response was linear and therefore Delta Cpo = 0. Enthalpy and entropy changes were calculated for the indicated buffer and protein concentrations, and therefore named as Delta Hobso and Delta Sobso throughout the text.

To determine the steady-state fluorescence anisotropy, <r>, of phi 29 SSB, the corresponding vertically (Ipar ) and horizontally (Iperp ) polarized emission intensities elicited by vertically polarized excitation were corrected for background scattering and included in the described equations for L-format geometry fluorimeters. Thus, <r> was obtained from the ratio (Ipar  - GIperp )/(Ipar  + 2GIperp ). The constant G, accounting for the differential polarization sensitivity of the photodetection set-up, was estimated as described previously (36). The protein concentration (10-15 µM) used was low enough to prevent turbidity of the solution and, therefore, light scattering that could result in an artifactual depolarization of the fluorescence (28).

Time-resolved Fluorescence Spectroscopy

The decay of total fluorescence intensity, measured at a magic angle orientation of the polarizers, and those of the parallel and perpendicular components were recorded at 22 °C in a time-correlated single-photon counting spectrometer, as described (36), for a protein concentration ranging from 10 to 30 µM. The samples were excited at 290 ± 4 nm with vertically polarized light pulses from a thyratron-gated nanosecond flash lamp (Edimburg Instr. El 199) filled with N2. The emission was isolated with a cut-off 310 nm (Schott KV) and a band-pass U-54 (Schott) filters, and detected with a Phillips XP2020Q photomultiplier.

The kinetic parameters (lifetimes tau i, and amplitudes, alpha i) of the decay of the fluorescence intensity were determined by iterative convolution of the signal recorded at magic angle, I54(t). The fitting routines were based on non-linear least-squares techniques (37, 38), using standard weighting factors (39). Wavelength-dependent timing effects in the photodetection were corrected as indicated (36). The weighted average lifetime, <A><AC>&tgr;</AC><AC>¯</AC></A>, was computed from,
<A><AC>&tgr;</AC><AC>¯</AC></A> =<LIM><OP>∑</OP><LL>i</LL></LIM>&agr;<SUB>i</SUB>&tgr;<SUB>i</SUB> (Eq. 1)
The fluorescence quantum yield of phi 29 SSB was also estimated by time-resolved techniques as the ratio <A><AC>&tgr;</AC><AC>¯</AC></A>/tau R, where tau R stands for the radiative lifetime of tyrosine, 27 ± 2 ns (33, 40).

The fluorescence anisotropy decay parameters (rotational correlation times, phi i, and amplitudes, beta i) were determined by simultaneously fitting the measured intensities Ipar , Iperp to a sum of exponentials using a non-linear least-squares global analysis method as detailed elsewhere (36). The experimental decay of the tyrosine fluorescence anisotropy of phi 29 SSB was best fitted to a biexponential function,
r<SUB>o</SUB>(&bgr;<SUB>1</SUB>e<SUP><UP>−t</UP>/&phgr;<SUB>1</SUB></SUP>+&bgr;<SUB>2</SUB>e<SUP><UP>−</UP>t/&phgr;<SUB>2</SUB></SUP>), <LIM><OP>∑</OP></LIM>&bgr;<SUB>i</SUB>=1 (Eq. 2)
where ro is the anisotropy of the tyrosine emission at t = 0.

The decay of the fluorescence intensity and anisotropy contains subnanosecond components that were not well resolved in the flash lamp spectrometer. To have a better definition of these parameters, a set of both kinds of measurements were also carried out at the Laboratoire of Biochimie Moleculaire et Cellulaire (Orsay, Paris) with a Ti:sapphire (Spectra Physics) ps laser spectrometer, tuned at the third harmonic (287 nm) and with an instrument response function of 75 ps; the tyrosine emission was detected at 308 and 315 nm (Delta lambda  = 4 nm).

pH-dependent Tyrosine Ionization

To measure the tendency of the tyrosine phenolic hydroxyl groups of phi 29 SSB to undergo ionization, small amounts of a stock solution of 2.5 M NaOH were added to a solution containing the indicated concentrations of protein in buffer B, the change in volume at the end of the titration being <2.5%. The pH increase was recorded simultaneously with the decrease in the fluorescence at 308 nm. Free tyrosine was taken as a model since its fluorescent properties depending on the ionic environment in response to pH increase are well documented (41, 42, 43) and no equivalent data were, to our knowledge, available for NAcTyrA. Corrections for dilution and filter effects were made as described (21).


RESULTS

Theoretical Analysis Based on the phi 29 SSB Primary Structure

The amino acid sequence of phi 29 SSB (Fig. 1A) was analyzed with currently available computer programs to assess its basic theoretical structural features. According to Chou-Fasman secondary structure predictions (44), the region around tyrosines 50 and 57 (see Fig. 1A) showed a high probability of being in the protein surface. Moreover, analysis of the sequence-related hydropathy (45) indicated that this same region (from residues 49 to 69), based on the negative values of the Kyte-Doolittle index (Fig. 1B), could not be highly hydrophobic.


Fig. 1. Theoretical features of phi 29 SSB structure deduced from its primary sequence. A, the regions with a higher probability of being in the protein surface (Chou-Fasman predictions, Ref. 43) are boxed. The three tyrosines, at positions 50, 57, and 76 are indicated with asterisks. B, hydropathy profiles (Kyte-Doolittle index; 45) of phi 29 SSB. The location of residues limiting the regions with high hydrophilicity (shadowed) is indicated with numbers.
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In order to have a more reliable picture on both, the accessibility of the tyrosines of phi 29 SSB to the solvent, and potentially to DNA, as well as their ionic environment, we analyzed the parameters defining the steady-state and the time-resolved intrinsic fluorescence properties of the phi 29 SSB. The results obtained are summarized in Table I.

Table I.

Parameters of the tyrosine-dependent fluorescence of phi 29 SSB


Steady statea Time-resolvedb

 lambda excmax 276 nm  tau 1 (alpha 1) 0.4  ± 0.1 ns (0.39 ± 0.03)
 lambda emmax 308 nm  tau 2 (alpha 2) 1.5  ± 0.1 ns (0.27 ± 0.03)
 Phi FST 0.065 ± 0.010  tau 3 (alpha 3) 3.8  ± 0.1 ns (0.34 ± 0.03)
NE 3.1 ± 0.3  <A><AC>&tgr;</AC><AC>¯</AC></A> 1.8  ± 0.1 ns
Acryl  Phi FTR 0.067  ± 0.010
  kqc (3.82 ± 0.50) × 109 M-1 s-1  phi 1 triple-bond  phi short (beta 1) 0.14  ± 0.06 ns (0.24 ± 0.03)
  A 4 pi  phi 2 triple-bond  phi long (beta 2) 7.0  ± 0.5 ns (0.76 ± 0.03)
  facc 1 rod 0.28  ± 0.01
KI rinfinity e 0.21  ± 0.05
  kqc (2.33 ± 0.20) × 109 M-1 s-1
  A 3.3 pi  
  facc 1
 < r> 0.173 ± 0.01

a  All the measurements were done in buffer B at 25 °C.
b  These measurements were done in buffer A, at 25 °C for lambda exc 294 nm and lambda em 310 nm.
c  Effective quenching rate constant of the fluorescent tyrosines of phi 29 SSB. In the same experimental conditions, kq of Acryl and KI for NAcTyrA were 5.1 × 109 M-1 s-1, and 5.3 × 109 M-1 s-1, respectively (for these calculations, <A><AC>&tgr;</AC><AC>¯</AC></A> of NAcTyrA was considered as 1.6 ns; 52).
d  Anisotropy at t = 0.
e  Expected limiting anisotropy if the protein was immobile (see Equation 3).

Fluorescent Tyrosines of phi 29 SSB Are Not Ionized at Neutral pH

The fluorescence spectra of phi 29 SSB free in solution, pH 7.5, and complexed with ssDNA are depicted in Fig. 2. The area under the excitation and emission spectra is reduced by 90% upon binding, although the same excitation and emission maxima, lambda exc = 276 nm and lambda em = 308 nm, respectively, were observed in both cases. Moreover, the emission spectra did not show any shoulder around 340 nm and, therefore, ionization or strong hydrogen bonding of the phenol hydroxyl group of tyrosine does not seem to take place (28, 46) either for free or complexed phi 29 SSB.


Fig. 2. Corrected fluorescence spectra of phi 29 SSB. Data were obtained at a final concentration of 8 µM in buffer B at 25 °C in the absence (thick curves) and presence (thin curves) of saturating amounts of ssDNA (300 µM poly(dT). The excitation (lambda em = 308 nm) and emission spectra (lambda exc = 276 nm) were corrected for background emission, inner filter effect of DNA absorption and wavelength dependence of the fluorimeter response as indicated under "Materials and Methods."
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As expected for A-type proteins, that is, proteins lacking tryptophan in their primary structure (28, 47), no changes in either the shape of the excitation or the emission spectra were observed for the denatured phi 29 SSB (4 M guanidinium chloride) (not shown).

Fluorescence Emission of phi 29 SSB Is Polarized

The anisotropy of phi 29 SSB fluorescence monitored by continuous excitation with polarized light gave a value of 0.173 ± 0.010 (at 22 °C), much smaller than the maximum (ro = 0.32) corresponding to immobilized tyrosines (48, 49). Different extrinsic mechanisms (once discarded light scattering and other artifactual effects, see "Materials and Methods"), like radiationless energy transfer among the residues or rotational mobility of the fluorophore and/or the protein, could account for this depolarization. The former is very unlikely since it would require spectral overlap among the excitation and emission spectra that is not observed (Fig. 2) and a much higher concentration of the residues (28). To distinguish between localized motions of the tyrosine residues (that would be indicative of their rotational mobility) and global motions of the protein, the time-resolved fluorescence anisotropy of phi 29 SSB was analyzed.

The decay of the anisotropy was best fitted (chi 2 = 1.17) to biexponential functions (see Equation 2 and Fig. 3) with a ro value (0.28 ± 0.01) close to the maximum expected for the tyrosine chromophore (see above), and two well separated correlation times. The fast one, phi short = 0.14 ± 0.06 ns, accounts for approximately one-fourth of the depolarization. The remaining anisotropy decays almost completely through the slow rotational component, phi long = 7.0 ± 0.5 ns. Since phi short <<  phi long the decay of the total anisotropy can be expressed as a product of two depolarizing processes, one due to fast movements of the protein segment containing the tyrosine residues (r'(t)), and another related to the global rotational motion of the whole protein (for reviews, see Refs. 50 and 51), as,
r(t)=r′(t)e<SUP><UP>−</UP>t/&phgr;<SUB><UP>long</UP></SUB></SUP> (Eq. 3)
where r'(t) = (ro - rinfinity )e-t/phi short + rinfinity , rinfinity being the limiting anisotropy that would be attained if the tumbling of the whole protein was arrested. According to the parameters of the anisotropy fitting (Table I), rinfinity was found to be 0.21. 


Fig. 3. Time-resolved fluorescence anisotropy of phi 29 SSB. A solution of phi 29 SSB (12 µM) in buffer B was excited at 22 °C with a vertically polarized light pulse as indicated under "Materials and Methods." The vertical and horizontal components of tyrosine emission were detected at 308 nm as a function of time to compute the decay of the phi 29 SSB fluorescence anisotropy. The best fit (chi 2 = 1.17) to the experimental data was achieved for a biexponential function with phi 1 = 0.14 ± 0.06 ns (beta 1 = 0.24 ± 0.03) and phi 2 = 7.0 ± 0.5 (beta 2 = 0.76 ± 0.03). The residuals are shown in the bottom part of the figure.
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On the other hand, since,
&phgr;<SUP><UP>−</UP>1</SUP><SUB><UP>short</UP></SUB>=&phgr;<SUP><UP>−</UP>1</SUP><SUB><UP>segmental</UP></SUB>+&phgr;<SUP><UP>−</UP>1</SUP><SUB><UP>global</UP></SUB>; &phgr;<SUP><UP>−</UP>1</SUP><SUB><UP>long</UP></SUB>=&phgr;<SUP><UP>−</UP>1</SUP><SUB><UP>global</UP></SUB> (Eq. 4)
the short correlation time can be assigned to fast segmental motions of the tyrosine residues, and the long correlation time to the global motion of the entire protein (see Ref. 50 for review).

The Efficiency of the Fluorescence Emission of phi 29 SSB Is High

The efficiency of the tyrosine emission of phi 29 SSB was assessed by measuring the quantum yield of its global fluorescence emission in time-resolved (Phi FTR) and steady-state (Phi FST) measurements.

The experimental decay of the total protein fluorescence was best fitted (chi 2 = 1.1) to triexponential functions that gave the lifetimes tau 1 = 0.4 ± 0.1 ns, tau 2 = 1.5 ± 0.1 ns, and tau 3 = 3.8 ± 0.1 ns, the pre-exponential terms (amplitudes) being alpha 1 = 0.39 ± 0.03, alpha 2 = 0.27 ± 0.03, and alpha 3 = 0.34 ± 0.03, respectively. Since the fluorescence decay kinetics of polypeptides and proteins, even with a single tyrosyl residue, is usually complex (22, 46, 49), the results presented here should be taken as a numerical approximation to a more complex kinetics. However, the averaged lifetime of the protein fluorescence (<A><AC>&tgr;</AC><AC>¯</AC></A> = 1.8 ± 0.1 ns) is a valid parameter to estimate the global quantum yield of the fluorescence emission of phi 29 SSB. Thus, the ratio between and the radiative lifetime of the tyrosine tau R (see "Materials and Methods") gave a (Phi FTR) of 0.067 ± 0.010. A similar value (see Table I) was obtained when the quantum yield was estimated in steady-state measurements (Phi FST) when the area of the emission spectra of phi 29 SSB was compared to that of the reference emitter, the well characterized 5-MeOI (not shown). Therefore, the phi 29 SSB tyrosine-dependent fluorescence is not being quenched by ground-state mechanisms. In contrast to the situation found in most proteins, the value of Phi FST did not increase upon denaturation (results not shown).

The intensity of phi 29 SSB fluorescence was directly dependent on the temperature of the solution, this dependence being biphasic with a clear slope change at 45 °C (Fig. 4). This transition could reflect conformational changes in the environment of the fluorescent tyrosines, rather than on the global conformation of phi 29 SSB, which seems to be stable up to 50 °C (23).


Fig. 4. Steady-state analysis of the temperature effect on phi 29 SSB fluorescence emission. A solution containing 12 µM phi 29 SSB buffer B at 22 °C was excited (lambda exc = 276 nm) with a continuous light source (see "Materials and Methods"). Fluorescence emission (I54) was recorded at 308 nm with magic angle orientation of the polarizers (excitation 0°, emission 54°) to avoid anisotropic effects. Samples were kept 5 min at the indicated temperatures before carrying out the measurements. The estimated errors were <2%.
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All Tyrosine Residues of phi 29 SSB Are Fluorescent

If there is no energy transfer among the tyrosines (see below), it is possible to estimate whether or not a tyrosine(s) contributes appreciably to the observed emission by combining the estimated global Phi F and of the protein fluorescence. This is based on the fact that the pre-exponential terms of the function that fits the experimental fluorescence decay data are related to the fractional concentration of the emitting species. Thus, the number of emitting fluorophores (NE) can be related to that of the excited residues by the expression NE = NTPhi FST/(<A><AC>&tgr;</AC><AC>¯</AC></A>/tau R), where NT is the total number of fluorophores (25). In our case, NE was calculated to be ~3. Therefore, all tyrosines contribute to phi 29 SSB, although they may not be necessarily mechanistically identical.

Fluorescent Tyrosines of phi 29 SSB Are Equally Accessible to Solvent and Placed in an Exposed and Negatively Charged Microenvironment

It is well known (41, 42) that positive charges in the surroundings of a tyrosine facilitate ionization, allowing tyrosinate to appear below pH ~ 10, the pKa of the free tyrosine. On the contrary, in the presence of a negative environment or for buried tyrosines, a higher pH is required for effective ionization.

Ionization of phi 29 SSB tyrosines was followed by the extent of fluorescence quenching at 308 nm as a function of pH increase, since the quantum yield of tyrosine emission is highly reduced upon ionization (47). The results, compared with those of the free tyrosine are shown in Fig. 5. From pH 7.5 (the standard during the functional assays of phi 29 SSB) to pH 9, the quenching pattern of phi 29 SSB closely resembles that of the exposed tyrosine in solution. However, both ionization patterns became different at higher pH. The initial fluorescence was reduced by 50% at pH 10.2 for free tyrosine, as expected, and at pH 10.9 for phi 29 SSB. Therefore, either (i) the phi 29 SSB tyrosines are located in heterogeneous regions and they are differentially ionized at different pH or (ii) they are exposed in the protein surface, and as a consequence of denaturing effects became protected from ionization (by being included within the protein bulk and/or by being surrounded by negatively charged residues). More precise information could not be obtained by this technique, since the protein structure, as indicated by CD spectra, was significantly affected at pH > 9 (23). Further insight on the tyrosine microenvironment, in terms of solvent accessibility and neighbor ionic residues, was obtained by measuring the quenching of the protein fluorescence by small and highly diffusible quencher agents: acrylamide, iodide, and cesium ions.


Fig. 5. pH-dependent ionization of the phenol hydroxyl group of phi 29 SSB tyrosines. Fluorescence intensity as a function of progressive pH increase, recorded at lambda em = 308 nm, of a solution of phi 29 SSB (6 µM, closed symbols) compared with a solution of free tyrosine (5 µM, open symbols). A 50% reduction of the initial fluorescence was observed at pH 10.3 for free tyrosine and 10.9 for the tyrosyl residues of phi 29 SSB.
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Acrylamide, a highly efficient quencher of the tyrosine fluorescence (52), which does not interact with proteins and is not influenced by nearby charged residues (20, 31), was used to analyze the exposure of the emitting residues of phi 29 SSB to solvent. Stern-Volmer plots for the acrylamide quenching of phi 29 SSB fluorescence (Fig. 6A) were linearly dependent on the quencher concentration, the fractional accessibility being 1 (Fig. 6B). The rate constant of the global quenching process, kq,eff (acryl) was (3.8 ± 0.5) × 109 M-1 s-1, 75% of the value measured for the fully accessible NAcTyrA under the same conditions. This difference may be related to the difference in the diffusion coefficients of the tyrosyl residues of the protein relative to free NAcTyrA, since the solid angle Omega  under which the acrylamide can approach these fluorophores was estimated as 4pi for both, the set of tyrosyl residues of phi 29 SSB and the NAcTyrA. Therefore, these results are consistent with a situation in which the fluorescent tyrosines of phi 29 SSB are equally accessible to the solvent, and, apparently, located in non-buried regions of the protein.


Fig. 6. Stern-Volmer analysis of the solvent accessibility and ionic environment of the fluorescent tyrosines of phi 29 SSB. Fluorescence quenching of phi 29 SSB (11.7 µM, closed symbols) upon addition of increasing amounts of acrylamide (A) or KI (C) was compared to that of NAcTyrA (20.9 µM, open symbols). In both cases, solutions were made in buffer B and kept at 25 °C. The slopes of the plots (Ksv,eff) allow the calculation of the bimolecular rate constant, kq,eff, as indicated in the text. The fraction of tyrosines accessible to acrylamide (B) and I- (D) were obtained from the y axis intercept of double-reciprocal Lehrer-modified Stern-Volmer plots. The values of these parameters are listed in Table I.
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The ionic environment of the phi 29 SSB tyrosine residues was assessed by using I- (as KI) and Cs+ (as CsCl) as quenchers. Fig. 6C shows the results obtained in titrations of phi 29 SSB and NAcTyrA with KI. Again, the plots were linear, with all of the fluorophores being accessible (Fig. 6D). However, the quenching was less efficient, kq,eff (I-) being (2.3 ± 0.2) × 109 M-1 s-1, ~46% of NAcTyrA, and the angle Omega  = 3.3pi (see Table I). Thus, it seems that although all of the fluorescent tyrosines of phi 29 SSB are homogeneously quenched by I-, they seem to be somehow shielded by negative charges that would make the interaction with the quencher difficult. We also used a positively charged quencher (Cs+), but the results were erratic. Due to its low quenching efficiency (33), high amounts of salt (more than 0.6 M) had to be added to significantly quench the phi 29 SSB fluorescence, giving rise to important changes in the ionic strength of the solution and, most likely, in the protein organization. Similar undesired effects of CsCl have been previously reported for other A-type proteins, e.g. ribosomal proteins S8 and S15 (33) or calmodulin (52).

Temperature Has an Opposite Effect on the Thermodynamic Parameters K and omega  of phi 29 SSB-ssDNA Complex

Changes in the intensity of phi 29 SSB intrinsic fluorescence induced upon ssDNA interaction at different temperatures were studied to determine the relative contribution of the different thermodynamic parameters on phi 29 SSB-ssDNA complex formation. The experimental temperature range was 10-42 °C, where the protein conformation is stable (23) and the intensity of the fluorescence emission is linearly dependent on temperature (Fig. 4). Data from direct titrations of poly(dT) with increasing amounts of phi 29 SSB were processed according to the "unlimited" cooperative model of McGhee and von Hippel (35) as outlined under "Materials and Methods" (see Ref. 21, for further details). Representative examples of the experimental assays at 15 and 42 °C and the fitting procedure are shown in Fig. 7, A and B, respectively. The value of the nucleotide site size, n, the intrinsic affinity K, and the cooperativity, omega , are indicated in Table II. Qmax and n were found to be relatively independent on the temperature of the binding solution. On the contrary, K was highly affected by temperature increase, diminishing from 7100 M-1 (10 °C) to 700 M-1 (42 °C). Interestingly, the cooperativity parameter omega  increased with temperature, being ~4 times higher at 42 °C (omega  = 220) than at 10 °C (omega  = 56). The result of these compensating effects was a slight (2.5-fold) reduction of the effective DNA binding constant Komega . The van't Hoff plot of Komega versus 1/T was linear within the temperature range used (Fig. 8), indicating that Delta Cpo = 0 and, therefore, that the enthalpy (Delta Ho) is independent of temperature (53). Approximated values of the enthalpy (Delta Hobso) and the entropy (Delta Sobso) of the phi 29 SSB-ssDNA complex formation were obtained from the y axis intercept and the slope of the plot, respectively. Thus, Delta Hobso ~ -5 kcal mol-1 and Delta SobsoDelta  ~ 8 cal K-1 mol-1.


Fig. 7. Temperature effect on phi 29 SSB-ssDNA complex formation. A, direct titrations of poly(dT) with increasing amounts of phi 29 SSB at 15 °C (open symbols) and 42 °C (closed symbols). The broken line at the beginning of the titration represents the increase in fluorescence resulting from the absence of DNA binding. The emission constant of the free protein, fA, was estimated from the slope of this line. The same slope was achieved at the end of the titration, indicating that all of the present DNA has being saturated. B, Scatchard plot of the best fit (lines) of the experimental data (points) corresponding to the experiments shown in A. The fluorescence intensity at each protein concentration [SSB]T, together with the fA and Qmax, allowed the determination of the amount of free [SSB]F and bound [SSB]B protein. From these values, the binding density (nu  = [SSB]B/[DNA]) corresponding to the experimental data was estimated and compared with theoretical ones computed according to the McGhee and von Hippel's unlimited cooperative model for binding of proteins to large lattices (35) as described previously (21). The calculated values for the thermodynamic parameters of phi 29 SSB-ssDNA for these and other temperatures are shown in Table II.
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Table II.

Value of the thermodynamic parameters n (nucleotide site size), K (intrinsic DNA binding constant), omega  (cooperativity), and Komega (effective DNA binding constant) defining phi 29 SSB-ssDNA complex formation as a function of temperature


Temperature Thermodynamic parametersa
Qmax n K  omega Komega

°C nt m-1 105 m-1
10 0.90  ± 0.01 3.8  ± 0.1 7100  ± 120 56  ± 4 3.97  ± 0.20
15 0.92  ± 0.02 3.7  ± 0.3 5100  ± 610 62  ± 10 3.16  ± 0.50
20 0.97  ± 0.02 3.6  ± 0.2 4550  ± 120 63  ± 5 2.87  ± 0.20
25 0.95  ± 0.03 3.5  ± 0.2 3750  ± 200 71  ± 6 2.66  ± 0.10
30 0.96  ± 0.02 3.5  ± 0.1 2350  ± 150 83  ± 4 1.95  ± 0.20
37 0.97  ± 0.03 3.3  ± 0.3 1390  ± 160 140  ± 13 1.94  ± 0.60
42 0.95  ± 0.03 3.2  ± 0.2 700  ± 130 217  ± 8 1.52  ± 0.20

a  Theoretical values giving the best fit to experimental data corresponding to direct titrations of ssDNA (30-40 µM) in buffer B, analyzed according to the unlimited cooperative model of McGhee and von Hippel (35) as outlined before (see Ref. 21). The maximal quenching (Qmax) values upon complex formation were determined as (96 ± 3%) from dilution experiments (21, 66) carried out in the same conditions. Poly(dT) was chosen as a DNA binding lattice to ensure homogeneous binding and therefore to avoid undesired sequence-dependent effects (53).


Fig. 8. van't Hoff analysis of the effective DNA binding constant of phi 29 SSB-ssDNA. The calculated Komega values of phi 29 SSB-ssDNA complex (see Table II) were plotted against the inverse of the solution temperature (Kelvin). The best fit to the data was achieved for linear expressions, which gave Delta Hobso and Delta SobsoDelta from the y axis intercept (as -Delta Hobso/R) and from the slope (as Delta SobsoDelta /R), respectively. In this case, Delta Hobso was -5 kcal mol-1, and Delta Sobso was ~8 cal K-1 mol-1. It should be recalled that these values are apparent, corresponding to the indicated protein concentration and buffer ionic conditions.
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DISCUSSION

SSBs share two main functional properties: several orders of magnitude more efficient binding to ssDNA than to dsDNA, and critical roles in the maintenance and expression of the genetic information. B. subtilis phage phi 29 encodes its own SSB, which is absolutely required for viral DNA replication in vivo. phi 29 SSB plays its role(s) by binding to the ssDNA regions of the replicative intermediates produced during strand-displacement phi 29 DNA replication (18, 19, 20). Therefore, the molecular basis of the contacts responsible for the formation and stability of this complex must be determined to properly understand the functional significance of phi 29 SSB during phage phi 29 infective cycle.

Towards the Identification of Residues Involved in Complex Formation

Steady-state fluorescence studies have served to determine that the interaction between phi 29 SSB and ssDNA is characterized by an unlimited cooperative DNA binding mode (omega  = 50-80 at 25 °C), a relatively low binding affinity (Keff = 1-3 × 105 M-1 at 25 °C), a small nucleotide binding size (n = 3-4 nt), and a net release of a very low number of cations (21). The lack of a clear sequence homology or defined patterns in protein motifs involved in ssDNA binding made difficult the identification of the phi 29 SSB residues interacting with ssDNA. One feature of the phi 29 SSB-DNA interaction that could give a hint on a potential DNA binding domain is the high reduction of the intrinsic tyrosine fluorescence of phi 29 SSB upon complex formation, the maximal quenching (Qmax > 95%) being one of the highest values reported for a SSB-ssDNA interaction (21). Among the several mechanisms described to account for tyrosine quenching in proteins that bind to DNA (22, 46, 54), those leading to the highest reduction in fluorescence intensity have been related to direct interactions between tyrosines and DNA (22, 46). It should be kept in mind that since phi 29 SSB binding is cooperative, the possibility that quenching occurs as a consequence of protein-protein interactions must be also taken into account. In any case, a detailed knowledge of the tyrosine environment and accessibility in the phi 29 SSB molecule is necessary to understand the molecular nature of the interactions that contribute to phi 29 SSB-DNA complex formation.

The average quantum yield Phi F of the intrinsinc phi 29 SSB fluorescence emission, analyzed by steady-state and time-resolved measurements, gave a value of 0.065 ± 0.010. The fact that both techniques gave the same Phi F value can be considered as indicating that none of the three possible emitters has been eliminated by ground-state deactivating processes. In other words, the three tyrosines of phi 29 SSB contribute to its intrinsic fluorescence. This Phi F value is unusually high for A-type proteins, namely proteins that lack tryptophan in their primary sequence. Thus, values of 0.037, 0.016, and 0.001 have been reported for insulin, octopus calmodulin, and ovomucoid protein, respectively (47, 55). The main mechanism leading to tyrosine quenching in A-type proteins is related to peptide bond formation, and additionally, to interactions with the side chains of neighboring residues, which can act as partners in hydrogen bond formation (46, 56). Phi F of phi 29 SSB is ~70% of that of N-acetyl-L-tyrosine amide, the closest analogous of tyrosines included in polypeptidic bonds. Therefore, phi 29 SSB tyrosines do not appear to maintain strong interactions with other residues of the protein since this would lead to a much reduced value of the efficiency of fluorescence emission (56). In addition, the excitation and emission maxima of phi 29 SSB fluorescence at neutral pH (see Table I) are indicative of a lack of surrounding interactions leading to tyrosinate formation. An opposite situation has been described for the SSBs of the filamentous phages Pf1, Pf3 (57).

The Stern-Volmer analysis of the quenching by acrylamide and KI allowed us to propose that the fluorescent tyrosines are equally accessible to small quenchers and, likely, they are placed in an accessible domain of the protein. The kq,eff(acryl) for phi 29 SSB is (3.8 ± 0.5) × 109 M-1 s-1, similar to those reported for accessible tryptophan residues of E. coli SSB (58) and T4 gp32 (59). Moreover, the value of Omega , the solid angle related to the accessibility of tyrosines to acrylamide, together with the fractional fluorescence accessible to the quencher, 4pi and 1 for phi 29 SSB, respectively, correspond to those expected for totally accessible residues. The differences in the accessibility to a negatively charged quencher, iodide ion, kq,eff(IK) (2.3 ± 0.2) × 109 M-1 s-1, indicate that fluorescent tyrosines should have a net negatively charged microenvironment, in agreement with the pH dependence of tyrosine fluorescence of phi 29 SSB. The absence of upward curvature for both, acrylamide and iodide, could be interpreted as due to a dynamic quenching process (31), the contribution of a static quenching being negligible, at least for the range of quencher concentrations used in this work. This linear behavior is characteristic of a relatively homogeneous population of the fluorescence emitters (30). Although the fluorescent tyrosines could be located in different exposed regions of the protein, it would be rather unlikely that all of these domains have a similar ionic environment. Therefore, it is tempting to speculate that phi 29 SSB tyrosines could be located within the same solvent-accessible protein region.

Further information regarding the environment of the phi 29 SSB tyrosines was obtained from the analysis of the rotational depolarization of the intrinsic protein fluorescence. As shown above, time-resolved anisotropy revealed two depolarizing motions with clearly different correlation times. In the absence of resonance energy transfer, the short correlation time, phi short = 0.14 ± 0.06 ns, can be assigned to fast localized motions of the tyrosine side chains. This value is very close to that of free tyrosines in solution (0.13 ns; 60) and, therefore, can be considered as indicating absence of restrictive interactions among tyrosine side chains and other residues. The long correlation time (phi long = 7.0 ± 0.5 ns) describes a slower motion of the whole protein. According to the parameters of the anisotropy fitting (Table I), the limiting anisotropy, rinfinity , that would be attained if the tumbling of the whole protein was arrested, was found to be 0.21. From this value, the average angular oscillations of the emitting residues of phi 29 SSB were estimated (50) as spanning about 50 °C. In summary, phi 29 SSB tyrosines are able to undergo fast subnanosecond oscillations as if they were not blocked, although some steric hindrance, likely due to the presence of the rest of the protein bulk, partially limits the amplitude of these motions.

Thermodynamic Aspects of phi 29 SSB-ssDNA Complex

The large quenching of the intrinsic fluorescence of phi 29 SSB upon complex formation (Qmax = 95%; 21) precluded the analysis of the effect of DNA interaction on the rotational correlation times of the anisotropy, the lifetimes of the total fluorescence or the Stern-Volmer constants. However, the changes in the intrinsic fluorescence intensity as a result of DNA binding, monitored at different experimental temperatures, can be used to determine the individual contributions of the intrinsic affinity and the cooperativity parameters to the global binding process, as well as to obtain a rough estimation of the enthalpy and entropy values, as it has been described for other SSBs (52, 61, 62, 63). The increase of the reaction temperature in the range in which the global protein conformation is stable (see "Results" and Ref. 23) has opposite effects on the intrinsic binding constant, K, and on the cooperative parameter, omega , the effective binding constant (Keff = Komega ) being largely independent of temperature. Thus, while a ~10-fold decrease of K was estimated from 10 to 42 °C, an interesting simultaneous 4-fold increase is observed for omega  and, as a result, Keff is reduced only by ~2-fold. Therefore, thermally-induced brownian motions seem to favor a more accessible conformation of the protein domain involved in monomer-monomer interactions. The van't Hoff analysis of temperature effect on Komega allowed a gross estimation of the energies driving the global ssDNA binding process. The linearity of the van't Hoff plots, characteristic of non-site specific DNA interactions (53, 64) indicated that, at low salt concentration, Delta Hobso (~-5 kcal mol-1) was temperature independent (Delta CpobsoDelta  = 0), allowing the estimation of Delta SobsoDelta as ~8 cal K-1 mol-1.

Altogether, our results are consistent with fluorescent tyrosines being located either on the surface of the protein or in a pocket whose size and shape allow the quencher to gain access to the tyrosine residues. The fact that the nucleotide site size has a relatively small value (3-4 nt are covered by protein monomer; 21) supports the possibility of a discrete domain involved in complex formation with ssDNA. Furthermore, tyrosine mobility does not seem to be highly restricted by interactions with neighboring residues. This localization might contribute to complex formation, either indirectly, by establishing protein contacts between contiguous monomers, or through direct interactions with the DNA bases. Preliminary results support the last hypothesis, since phi 29 SSB point mutants in each of the three tyrosines have an impaired ability to bind ssDNA.2 In this sense, the presence of a net negative charge surrounding the tyrosines could be the reason for the relatively low affinity of this protein for ssDNA, a property which may be directly related to its functional roles during phi 29 DNA replication (20, 21). In addition, the relative independence of the effective DNA binding constant of phi 29 SSB of the solution temperature could provide an adaptative advantage for phage phi 29 development in the very different habitats, e.g. soil, field crops, different foodstuffs, seewater (see Ref. 65, for a review), in which the cellular host, B. subtilis, is able to grow.


FOOTNOTES

*   This work was supported in part by Grant 5RO1 GM 27242-16 from the National Institutes of Health, CHRX-CT93-0248 from the European Union, and PB93-0176 from DGICYT (to M. S.), and PB93-126 from DIGYCT and Acción Integrada Hispano-Francesa HF 94-183 (to A. U. A.). An institutional grant from Fundación Ramón Areces to Centro de Biología Molecular "Severo Ochoa" is also acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a predoctoral fellowship from Comunidad Autónoma de Madrid.
par    To whom correspondence should be addressed. Tel.: 1-397-8435; Fax: 1-397-4799.
1    The abbreviations used are: SSB(s), single-stranded DNA-binding protein(s); 5-MeOI, 5-methoxyindole; NAcTyrA, N-acetyl-L-tyrosine amide; nt, nucleotide(s); ssDNA, single-stranded DNA; Pf3 phage, Pseudomonas aeruginosa filamentous bacteriophage.
2    M. S. Soengas, unpublished data.

Acknowledgments

We are indebted to J. M. Lázaro and L. Villar (Centro Biología Molecular "Severo Ochoa", Madrid) for the purification of phi 29 SSB and Drs. M. P. Lillo and R. Dale (Instituto de Química Física Rocasolano, Madrid) for their invaluable contribution in data analysis and interpretation. We also thank Drs. J. C. Brochon and P. Tauc (Laboratoire of Biochimie Moleculaire et Celulaire. CNRS, Orsay, Paris) for making available to us the picosecond spectrometer and for their kind help with the measurements.


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