Nucleic Acid Binding Affinity of fd Gene 5 Protein in the Cooperative Binding Mode*

A sensitive ESR method which allows a direct quan- titative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd. This was achieved with two spin-labeled nucleic acids, (ZdT,dT), and (ZA,A)n, which served as macromolecular spin probes in ESR competition experi- ments. With the two different macromolecular spin probes, it was possible to determine the relative apparent affinity constants, Kapp, over a large affinity do- main. In 20 mM Tris. HC1 (pH S.l), l mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Tri- ton, and 125 mM NaCl, the following affinity relationship was observed: K::;). 1.5 10’ Increasing the [NaCI] from 125 to 200 mM caused considerably less tight binding of gene 5 protein to (ZA,A)n, and a typical cooperative binding isotherm was observed, whereas at the lower [NaCl] used for the competition experiments, the binding was essentially stoichiometric. A computer fit of the experimental titration data at 200 mM NaCl gave an intrinsic binding constant, Kin,, of 1300 M” and a cooperativity factor, W, of 60 (Kinrw = Kapp) for (zAA)n-The computer control of scan range, scan rate, time averaging of data, and ESR recorder operation. Each 100 G spectrum consists of 1000 data points at a digital resolution of 12 bits and the digitized spectral arrays are stored on floppy discs. For the titration and competition studies, an E-238 cavity was used along with an E-238-3 quartz flat ESR cell onto which a paramagnetic Cr3+ standard was externally fixed. ESR Data Analysis-The ESR spectra were analyzed in terms of a two-state model using a published algorithm (19). The program to determine the fraction of saturation, F, of the macromolecular spin probe is now written in BASIC and the analysis can be done directly with the Apple I1 plus. The paramagnetic Cr3+ standard to align the spectra was made by first pJeparing pure magnesium oxide which then was dissolved in dilute HCI solution. To this solution, a known amount of chromium nitrate was added and subsequently the solution was precipitated with ammonium hydroxide. After washing the precipitate, it was dried at 400 "C to yield magnesium oxide containing chromium as lattice impurity. This mixture was mixed with polyeth-ylene powder in different proportions and hot-rolled between Teflon sheets to give a Cr3+ standard which is stable and convenient to handle.


Kapp) for (zAA)n-
The gene 5 protein product of bacteriophage fd is essential for DNA replication during the life cycle of fd phage in Escherichia coli (for a review, see Refs. 1 and 2). Proposed structures for the fd DNA-gene 5 protein complex (3) and oligonucleotide-gene 5 protein complex (4, 5) recently have been given. By a variety of spectroscopic techniques, it was established that the gene 5 protein monomer binds to four nucleotide bases along the DNA lattice (6)(7)(8)(9). The protein has a molecular weight of 9690 and exists primarily as a dimer in neutral buffers which are 0.15 M in NaCl (10, 11). It binds to single-stranded DNA of any sequence, and binding studies with defined oligodeoxynucleotides suggest that it has a much greater affinity for adenine-rich regions than for thyminerich regions (8). Binding of the protein to single-stranded DNA is cooperative (12), and it was also shown that it forms a complex with an isolated tetranucleotide with a dissociation constant of M, whereas the complex with fd DNA has a dissociation constant of -lo-' M (7). With respect to interaction specificity, it has often been tacitly assumed that oligonucleotide binding to gene 5 protein approximates polynucleotide binding, although the cooperative protein-protein * This work was supported in part by National Institutes of Health Grant GM-27002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact,.
1: To whom correspondence should be addressed.
interactions may play an important role in binding specificity.
The high binding affinities often present in protein-nucleic acid interactions have caused serious problems when analyzing such interactions by conventional physical methods. In order to better understand the fundamental recognition process in molecular biology, it is necessary to design experimental methods which allow reliable characterization of binding specificity under physiological conditions. The DNase protection ("footprinting") method applied recently for estimating differential binding constants (13,14) allows one to readily discriminate sequence specificity (differential binding constant) within at least one order of magnitude (13) by making some straightforward approximations for analyzing the digestion patterns. From data based on the DNase protection experiments, values for the cooperative interaction of the bacteriophage X repressor with the X right operator were determined which allowed the development of a model for gene regulation by the X phage repressor (15). This laboratory put considerable effort in designing a sensitive ESR approach which allows a direct quantitative determination of relative nucleic acid binding affinities of proteins binding under physiologically relevant conditions to nucleic acids. The approach is based on competition experiments with macromolecular spin probes such as (ldT,dT),' and allows one to distinguish between small as well as large relative nucleic acid binding affinities. We had already shown qualitatively in 1975 that, for instance, the bacteriophage T4 gene 32 protein has a much greater affinity for (dT), than for (a), (16). More recently, we described the quantitative ESR approach for determining the relative affinity of the gene 32 protein for various polynucleotides (9). Here, we use two macromolecular spin probes, (ldT,dT), and (lA,A),, to determine the relative binding affinity of gene 5 protein for various polynucleotides. Our results confirm some qualitative observations made earlier with respect to the binding of gene 5 protein to fd DNA and R17 RNA (17), but we could find no evidence for the general conclusion that gene 5 protein has substantially greater affinity for adenine containing nucleotides than for thymine containing nucleotides (8).  http://www.jbc.org/ Downloaded from light scattering (6). All homopolymers were purchased from P-L Biochemicals, whereas fd DNA and a mixture of (40%) 16 S + (60%) 23 S ribosomal RNA were bought from Miles Laboratories, Inc. R17 RNA was prepared and purified as described earlier (18).

Gene 5 protein was a generous gift of Loren
Preparation of Spin-labeled Polynucleotides-( ldT,dT),, (lA,A),, and (IdA,dA), were prepared by chemical modification of (dT), and (A)n, respectively, with 4-(cu-iodoacetamido)-2,2,6,6-tetramethylpiperidino-1-oxy (Eastman Organic Chemicals) by using a previously published procedure. The spin-labeled and unlabeled polynucleotides were all purified through Sephacryl S-200 (1.5 X 80 cm) eluted with 0.04 M ammonium bicarbonate. Disc gel electrophoresis according to standard procedures was used to check the homogeneity and weightaverage molecular weight of the nucleic acids. All spin-labeled and unlabeled synthetic polynucleotides had molecular weights of 100,000-200,000. The nitroxide-labeled nucleotide to unlabeled nucleotide ratio is given in the figure legend and was determined according to standard procedures.
Nucleic Acid Concentrations-Nucleic acid concentrations were calculated from solution absorbances with the following extinction coefficients (X10-3 M" cm"): .~~ ESR Measurements-All ESR sDectra were obtained with a Varian E-104.4 Century Series Spectrometer which was interfaced with an Apple I1 plus microcomputer. Customized software was developed in our laboratory to provide for computer control of scan range, scan rate, time averaging of data, and ESR recorder operation. Each 100 G spectrum consists of 1000 data points at a digital resolution of 12 bits and the digitized spectral arrays are stored on floppy discs. For the titration and competition studies, an E-238 cavity was used along with an E-238-3 quartz flat ESR cell onto which a paramagnetic Cr3+ standard was externally fixed.
ESR Data Analysis-The ESR spectra were analyzed in terms of a two-state model using a published algorithm (19). The program to determine the fraction of saturation, F, of the macromolecular spin probe is now written in BASIC and the analysis can be done directly with the Apple I1 plus. The paramagnetic Cr3+ standard to align the spectra was made by first pJeparing pure magnesium oxide which then was dissolved in dilute HCI solution. To this solution, a known amount of chromium nitrate was added and subsequently the solution was precipitated with ammonium hydroxide. After washing the precipitate, it was dried at 400 "C to yield magnesium oxide containing chromium as lattice impurity. This mixture was mixed with polyethylene powder in different proportions and hot-rolled between Teflon sheets to give a Cr3+ standard which is stable and convenient to handle.

RESULTS
We reported earlier the ESR line shapes of (IdT,dT), in the presence and absence of gene 5 protein (9) as well as plots of F, the fraction of complexed (ldT,dT), versus [protein]/ [dT] based on ESR titrations of the macromolecular spin probe (ldT,dT), with the protein. In Fig. 1, we show the line shape chhnge of (IA,A), in the absence and presence of gene 5 protein. As in the case of (IdT,dT),, the binding of the protein ligand to (IA,A), gives rise to a significant change in the ESR line shape. Complexation broadens the spectrum considerably, and the overall line shapes of the (IdT,dT),. gene 5 protein complex (9) and the (IA,A),. gene 5 protein complex look similar. The substantial difference between spectral array A and B in Fig. 1 allows the monitoring of the binding of the gene 5 protein to (lA,A),,. By utilizing the entire digitized ESR spectral arrays measured during the titration of (lA,A), with gene 5 protein, it was found that a twocomponent analysis method can be used to determine the fraction F of complexed spin-labeled polynucleotides as was the case for bacteriophage T4 gene 32 protein and (IdT,dT), or gene 5 protein and (ldT,dT), (9).
A typical analysis of titration results obtained with gene 5 protein and (lA,A), is shown in Fig. 2   of 50 and 125 mM NaCl) the binding is essentially stoichiometric, which allows one to determine the binding stoichiometry, since virtually all the protein added is bound. For that purpose, the initial slope which is the same for both salt concentrations is extrapolated to F = 1, and the projected value of the abscissa is taken for the [protein]/[A] ratio. It follows that gene 5 protein covers about 4 nucleotide residues, a value which is in good agreement with titration results obtained earlier with (IdT,dT), as well as with other spectroscopic techniques. At a higher salt concentration (e.g. in the presence of 200 mM NaCl), binding becomes less tight and a typical "cooperative" binding isotherm is observed. This binding isotherm is characterized by a lag phase at low protein concentration, where very little binding is seen, followed by a sharp rise in binding. It is apparent from Fig. 2 that the high binding affinity of gene 5 protein at 50 and 125 mM NaCl formalism developed recently for gene 32 protein (9) can be applied. As pointed out earlier, the experiments are conveniently carried out by first establishing the relationship existing between F and the empirical peak height ratio h,Jho for a particular protein-macromolecular spin probe system. In Fig.  4, this relationship is shown for gene 5 protein and (lA,A), or (ldT,dT),. It is apparent that the empirical peak height ratio changes considerably for both macromolecular spin probes as a function of lattice saturation. With Fig. 4, the fraction of saturated spin-labeled nucleic acid upon addition of some competitive ligands is readily determined by calculating the peak height ratios of the experimental ESR spectra measured in the course of competition experiments.
With Figs. 5 and 6, typical competition plots with the two macromolecular spin probes (lA,A), and (IdT,dT),, respectively, are shown with gene 5 protein as complexing ligand. Fig. 5 shows the effect of adding unlabeled nucleic acid competitors to (lA,A), complexed with gene 5 protein to reach an initial fraction of saturation, F,, between 0.64 and 0.74.   4. Plots of F, the fraction of complexed (lA,A), (1A.A =  0.03 2 0.003) (0) and (MT,dT), (IdTldT = 0.025 2 0.003) (H), uersus the h+/ho peak height ratios determined during the ESR titration with gene 5 protein under the buffer conditions described in Fig. 1 with 125  The competitions were done under the buffer conditions described in Fig. 1 with 125 Fig. 5 are 8 and 85, respectively, which gives a ratio KL$FNA/KL$) = 20 * 2, whereas considerably larger amounts of rRNA and R17 RNA were needed to "pull off" the gene 5 protein from the (IA,A), lattice. Using (e), as competitor would not have been meaningful, because the addition of gene 5 protein to the competition buffer solution containing (dA), or (ldA,dA), caused the formation of turbidity. With (IdT,dT), as macromolecular spin probe, it was possible to establish the relative affinity relationship between (dT), and fd DNA (Fig. 6). In that case, 9 nmol of (dT), and about 3000 nmol of fd DNA were needed to reach F, = 0. This results in a value of about 1000 for the KL$T)n/Ki$fNA ratio. Also, we established that fd DNA has no effect on the ESR spectra of (lA,A), and (ldT,dT), (data not shown) and therefore does not seem to interact with the two macromolecular spin probes. With the above shown competition data, the following affinity relationship was established 1.5 X IO5 KC;;RNA. Thus, under physiologically relevant conditions, (dT), displays an affinity for the gene 5 protein which is several orders of magnitude greater than that of fd DNA or (A),, and the data also demonstrate that (A), has a greater affinity for the protein than rRNA or R17 RNA.

DISCUSSION
The primary aim of this study was to apply the ESR competition assay to a different nucleic acid binding protein and to further expand the affinity range of the assay by using an additional macromolecular spin probe. The choice of the additional probe to evaluate quantitatively relative affinity constants was dictated by its protein affinity and ESR properties. In order to be useful, it had to display a considerably lower affinity for gene 5 protein than, for instance, (IdT,dT), and its ESR properties had to be such that the fraction of saturation of the probe could be conveniently monitored by using, for instance, some internal standardization as displayed in Fig. 4. Also, the new probe was expected, as the probe (IdT,dT),, not to interact with an unlabeled competitor such as fd DNA, so that fd DNA could be used to make crosscomparisons with results obtained using (1A,A), and (IdT,dT), as probes. We found that (lA,A), met all these criteria. On the other hand, both (dA), and (ldA,dA), could not be included in this study because they formed a precipitate with gene 5 protein in the competition buffer. No such precipitation was noted with all other nucleic acids. The reason for the observed precipitation is presently not known, and we noticed no such problems with (dA), or ( Z d A , d A ) , and gene 32 protein.
The spin assay to determine the affinity of nucleic acid binding proteins offers many advantages as outlined earlier (9, 22). Binding can be observed directly in solution under physiological conditions without isolating the protein-nucleic acid complex as is necessary with centrifugation or radiolabeling approaches. The measurements can be carried out with nanomole quantities of proteins and nucleic acids, which is considerably less than that required by other direct methods the protein for the macromolecular probe does not affect the Ktpp/K:pp ratio as follows from the derivation of Equation (9).
Thus, any potential perturbation introduced by the nitroxide radical in the macromolecular probe will not affect the relative affinity of the unlabeled nucleic acid competitors for the protein.
Although valuable information can usually be obtained in simple model systems by determining some parameters as a function of salt concentration or temperature, it is often necessary to make assumptions which might not be valid with macromolecules such as proteins and nucleic acids. For instance, there is some uncertainty about the ionic strength effect on the dimer formation of gene 5 protein, and therefore it would be extremely difficult to determine some binding characteristics with a ligand which changes its state as a function of salt concentration. It has been claimed that gene 5 protein remains a dimer under various conditions such as changing the ionic strength of the solution from 0 to 0.5 M KC1 or changing the pH from 5.0 to 11.0 or varying the temperature from 5 to 20 "C (11). On the other hand, it has been shown (10) that the monomer-dimer equilibrium is a strong function of salt type and concentration. It was noted that increasing the [NaCl] beyond 0.15 M had a definite effect on the sedimentation behavior of gene 5 protein, and it was suggested that the transition from dimers to monomers changes neither the CD nor the tyrosyl fluorescence of the protein (10). The observed decrease of binding affinity of gene 5 protein for the macromolecular probe (lA,A),, when increasing the ionic strength from 125 to 200 mM NaC1, could well reflect a shift in the monomer-dimer equilibrium, which in that case would mean an increased monomer concentration. It is reasonable to assume that, in the absence of unfavorable entropic or steric constraints, a gene 5 dimer would display greater affinity for nucleic acids than the monomer as was shown to be the case for bis(methidium)spermine (23). The absolute KaPp calculated for (lA,A), in 200 mM NaCl with two different formalisms was in the order of lo5 M" and w was 60. It is noteworthy that, based on sucrose gradient experiments and a straightforward simple model, it was already suggested in 1972 that the affinity of gene 5 protein for a contiguous site must be at least sixty times greater than its affinity for an isolated site (12).
In view of the above given monomer-dimer equilibrium problem, no attempt was made to systematically study the binding process with gene 5 protein as a function of salt concentration and to interpret the salt effects in terms of ionic interactions and release of structured water involved in the binding process (24, 25). We selected a NaCl concentration for the competition studies which results in very tight binding SO that the approximation of no free protein in solution made for the derivation of the competition formalism (9) is valid with both macromolecular spin probes. The affinity relationship observed under these tight binding conditions reveals, as in the case of gene 32 protein,differences in binding affinities of several orders of magnitude. Both proteins have by far the largest affinity for (dT),, and significant differences in affinity exist for all other nucleic acids tested.