Global Conformations, Hydrodynamics, and X-ray Scattering Properties of Taq and Escherichia coli DNA Polymerases in Solution*

Allison M. Joubert {ddagger}, Angela S. Byrd § and Vince J. LiCata 

From the Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

Received for publication, February 28, 2003 , and in revised form, April 22, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Escherichia coli polymerase 1 (Pol 1) and Thermus aquaticus Taq polymerase are homologous Type I DNA polymerases, each comprised of a polymerase domain, a proofreading domain (inactive in Taq), and a 5' nuclease domain. "Klenow" and "Klentaq" are the large fragments of Pol 1 and Taq and are functional polymerases lacking the 5' nuclease domain. In the available crystal structures of full-length Taq, the 5' nuclease domain is positioned in two different orientations: in one structure, it is extended out into solution, whereas in the other, it is folded up against the polymerase domain in a more compact structure. Analytical ultracentrifugation experiments report s20,w values of 5.05 for Taq, 4.1 for Klentaq, 5.3 for E. coli Pol 1, and 4.6 for Klenow. Measured partial specific volumes are all quite similar, indicating no significant differences in packing density between the mesophilic and thermophilic proteins. Small angle x-ray scattering studies report radii of gyration of 38.3 Å for Taq, 30.7 Å for Klentaq, and 30.5 Å for Klenow. The hydrodynamic and x-ray scattering properties of the polymerases were also calculated directly from the different crystal structures using the programs HYDROPRO (Garcia De La Torre, J., Huertas, M. L., and Carrasco, B. (2000) Biophys J. 78, 719–730) and CRYSOL (Svergun, D. I., Barberato, C., and Koch, M. H. J. (1995) J. Appl. Crystalogr. 28, 768–773), respectively. The combined experimental and computational characterizations indicate that the full-length polymerases in solution are in a conformation where the 5' nuclease domain is extended into solution. Further, the radius of gyration, and hence the global conformation of Taq polymerase, is not altered by the binding of either matched primer template DNA or ddATP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Understanding the conformation of an enzyme in solution is an essential aspect of the comprehensive characterization of the overall structure and function of that enzyme. Taq DNA polymerase is a single polypeptide chain consisting of three structure/function domains: a polymerase domain, a 3' exonuclease domain (inactive in Taq), and a 5' nuclease domain (15). The polymerase and inactive 3' exonuclease domains together comprise the "Klentaq" domain (6) (by analogy to the Klenow fragment of Escherichia coli). The spatial positioning of the 5' nuclease domain of Taq DNA polymerase has been the subject of debate, and crystal structures of the full-length protein have been reported with the 5' nuclease domain in two different positions relative to the Klentaq domain (5, 7, 8). The two structures are shown in Fig. 1. In one structure, the 5' nuclease domain extends directly out into solution, whereas in the other, this domain is folded up against the Klentaq domain.



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FIG. 1.
Alternate x-ray crystal structures of full-length Taq DNA polymerase (5, 8). The Klentaq domains (light gray) of the two structures are equivalent, but the position of the 5' nuclease domain (black) differs significantly.

 

The two reported conformations for Taq polymerase have potentially different functional consequences. In the elongated conformation of the polymerase, the polymerization and 5' nuclease active sites are separated by ~70 Å (5, 8). In order for a single polymerase molecule to simultaneously catalyze nucleotide incorporation and 5' nuclease activities in close proximity on the same piece of DNA, the polymerase would need to adopt a more compact conformation that would bring the two active sites into relative proximity (8). Although the compact conformation observed in the Urs et al. crystal structure is not ideally oriented for such simultaneous catalysis, the two active sites are in closer proximity than in the extended structure (8). The larger separation between active sites in the elongated conformation of the polymerase does not preclude binding of the same DNA to the two active sites; however, it would involve more distant spacing along the DNA between the two active sites. It is also certainly possible that under certain conditions, the polymerase can switch between elongated and compact conformations.

Analytical ultracentrifugation and small angle x-ray scattering provide different but complementary information on the size and shape of macromolecules in solution. The structural information provided by these techniques is quite "low resolution," but for large proteins, where NMR methods are only in their infancy, these techniques often provide some of the only solution structural information about a protein. In this study, we have characterized basic hydrodynamic properties (s20,w and partial specific volume) and x-ray scattering properties (radius of gyration) of full-length Taq and E. coli Pol 11 DNA polymerases and their Klentaq and Klenow subfragments. We have also performed structure-based calculations of these same parameters using software developed by Garcia de la Torre et al. (for hydrodynamic parameters) (9) and by Svergun et al. (for x-ray scattering parameters) (10). In addition to presenting the basic hydrodynamic and x-ray scattering properties of these proteins, we show that the data indicate that in the absence of DNA, both full-length Taq and full-length Pol 1 are in an elongated conformation in solution with their 5' nuclease domains sticking out into solution. Additionally, when bound to a matched primer template piece of DNA or to ddATP, Taq polymerase remains in an elongated conformation.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Materials—Polymerases were expressed and purified as described previously (11, 12). No surfactants were used during purification, experiments, or storage of the polymerases. Taq and Klentaq polymerases were stored at 4 °C before use. Pol 1 and Klenow polymerases were stored frozen at –20 °C (13). The DNA used for small angle x-ray scattering (SAXS) measurements of the Taq + DNA complex was a matched 13-/20-mer primer template pair with the sequence: 5'-TCGCAGCCGTCCA-3' paired with 3'-AGCGTCGGCAGGTTCCCAAA-5' as used previously for Taq polymerase DNA binding studies (12).

Sedimentation Coefficient Measurements—Sedimentation velocity experiments were performed in a Beckman Optima XL-A analytical Ultracentrifuge. Sedimentation coefficients were measured at 20 °C in KD buffer (10 mM Tris, 125 mM KCl, and 5 mM MgCl2, pH 7.9). The reference and sample sectors of Epon charcoal-filled double-sector cells were loaded with 425 µl of buffer and 400 µl of protein solution, respectively. All velocity runs were performed at 38,000 rpm in an An-60 Ti rotor for 3.5 h. The absorbance was monitored at 280 nm. Twenty absorbance scans with a 0.004-cm step size were recorded at 10-min intervals. Svedberg constants were determined from fits of the data to single ideal species using the program Svedberg (14, 15). All data were well fit by a single ideal species model across a range of concentrations. Taq concentrations examined ranged from 0.25 to 1.0 mg/ml. Klentaq concentrations examined ranged from 0.3 to 1.23 mg/ml. Klenow concentrations examined ranged from 0.36 to 1.25 mg/ml. Pol 1 was examined at 0.2 mg/ml only. All s values reported herein have been converted to s20,w values using measured solvent densities and viscosities.

Sedimentation Equilibrium Experiments and Partial Specific Volume Determination—The partial specific volumes of the polymerases were measured using the method of Edelstein and Schachman (16). Sedimentation equilibrium experiments were performed either in H2O/KD buffer or in 96% D2O/KD buffer. Experiments were performed in the Beckman Optima XL-A using the same rotor and cells used in the sedimentation velocity runs. The double sector cells were loaded with 125 µl of buffer and 110 µl of protein solution in matching buffer. Equilibrium runs were performed at 9500 rpm for Pol 1 and Taq and 11,000 rpm for Klenow and Klentaq. All runs were carried out at 20 °C for ~24 h or until equilibrium was reached. The absorbance was monitored at 280 nm, and the initial absorbance of each protein solution was between 0.1 and 0.6. Equilibrium data were analyzed using the Origin Equilibrium analysis package provided with the instrument. The partial specific volume of each polymerase in KD buffer at 20 °C was also calculated from the amino acid sequence using the computer program SEDNTERP (freeware, archived at www.bbri.org/rasmb/rasmb.html).

Density and Viscosity Measurements—Buffer density was measured at 20 °C using an Anton-Paar DMA 58 digital densitometer. A calibrated Cannon-Manning semimicro kinematic viscometer was used to measure the buffer viscosity. The measured efflux time was converted to viscosity using the equation, viscosity = t*V*{rho}, where t = efflux time, V = viscometer constant (in mm2s2) at temperature T (as supplied by the manufacturer), and {rho} = buffer density.

Small Angle X-ray Scattering—SAXS experiments were conducted on synchrotron beamlines 1-4 and 4-2 at the Stanford Synchrotron Radiation Research Laboratory (SSRL). Preliminary data were also collected on beamline D11A-SAXS at the Laboratorio Nacional de Luz Sincrotron (LNLS) in Campinas, Brazil. All data shown in the manuscript are from the SSRL beamlines. Data from SSRL beamline 4-2 were collected at a wavelength of 1.385 Å and sample to detector distances of 1.28 and 2.0 m. Data from SSRL beamline 1-4 were collected at a wavelength of 1.488 Å and a sample to detector distance of 0.38 m (which is optimal for this beamline). The beam flux was 2 x 1010 photons/s. The sample cell was aluminum and consisted of a 200-µl flat sided, round sample chamber. The path length through the sample was 1.4 mm and was bounded on both sides by circular Kapton windows. SAXS measurements were conducted for all four polymerases in KD buffer. Scattering was monitored in 15-min exposure times for each polymerase. Repeated measurements of the same sample yielded the same results, indicating that no significant radiation damage occurred during the experiments. Polymerase concentrations ranged from 0.9 to 5.3 mg/ml, and at least three different concentrations were examined for each protein. Determined Rg values did not show any protein concentration dependence within error. The scattering data were collected and normalized for dark counts and scattering intensity, and the buffer background scattering was subtracted using the resident analysis programs at each beamline.

SAXS experiments were conducted at very low protein concentrations for this technique. This was due to precipitation of the polymerases at higher concentrations. For example, full-length Taq polymerase will sometimes begin to precipitate at concentrations above 1.5 mg/ml. Lower protein concentrations lead to higher noise levels in the data and generally preclude detailed shape analysis of the data at higher q values. SAXS data were analyzed in three major ways: 1) The data were analyzed using Guinier plots (17) where Rg values were determined from the linear portions of the plots. 2) The data were analyzed using the program GNOM, where an indirect Fourier transform of the experimental scattering curve is implemented to derive the P(r) distance distribution function. The Rg is then calculated from the P(r) function (18). Program default values were used for all input parameters except for Dmax, which is altered to obtain the best fit (18). 3) The data were analyzed using the program CRYSOL, which fits the measured scattering curve with a simulated scattering curve generated using a known x-ray crystal structure (10). Some, but not all, of the scattering curves exhibited a sharp increased slope at very low q values (q < 0.02 Å1), which was removed before analysis. This behavior was only present in some of the beamline configurations used and seemed to be best explained as a beam stop problem (for example, none of the data shown in Figs. 5, 6, 7 exhibited this behavior). This behavior could not be attributed to aggregation since 1) it was sometimes absent; 2) it was generally identical from protein to protein, including control proteins such as aspartate transcarbamylase (data not shown); and 3) it was not correlated with the protein concentration. Furthermore, the equilibrium centrifugation experiments also conducted in this study showed no appreciable aggregation of the protein.



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FIG. 5.
Guinier plots (17) of the natural log of the small angle x-ray scattering intensity versus q2 for each of the DNA polymerases. Open circles show the data for full-length Taq, open triangles show data for Klentaq, diamonds show data for Klenow, and open squares show data for E. coli Pol 1. Pol 1 shows considerable curvature in the Guinier plot and so was not analyzed for an Rg value. The fitted lines used to determine the Rg values for Taq, Klentaq, and Klenow reported in Table II are shown. Protein concentrations are 5.1 mg/ml for Taq, 3.8 mg/ml for Klentaq, 6.3 mg/ml for E. coli Pol 1, and 5.3 mg/ml for Klenow. The data for Klentaq and Klenow are in the original ln (intensity) units. The data for Taq and Pol 1 have been displaced on the y axis by multiplying the original intensity values by factors of 0.25 and 10, respectively, for visual clarity.

 


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FIG. 6.
Fits of the log intensity versus q scattering profiles of Taq, Klentaq, and Klenow polymerases using the program GNOM (18). Inset graphs show the P(r) distance distribution function versus D for the best fit to each polymerase. Protein concentrations are 5.1 mg/ml for Taq, 3.8 mg/ml for Klentaq, and 5.3 mg/ml for Klenow.

 


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FIG. 7.
Correlations between crystal structure data and small angle scattering data. Fits of the experimental scattering data with simulated scattering data calculated from the two different crystal structures for full-length Taq polymerase (Taqa = 1TAQ [PDB] (5), Taqb = 1CMW [PDB] (8)), Klentaq polymerase (1KTQ [PDB] (19)), and Klenow polymerase (1KFD [PDB] (20)) were performed using the program CRYSOL (10). Open symbols show the experimental data, and solid lines show the fitted simulated scattering data calculated from the crystal structures. Protein concentrations are 5.1 mg/ml for Taq, 3.8 mg/ml for Klentaq, and 5.3 mg/ml for Klenow. Chi-squared ({chi}2) values for the goodness of fit were 3.78, 6.05, 3.74, and 3.23 for the fits to Taqa, Taqb, Klentaq, and Klenow, respectively.

 


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TABLE II
Partial specific volumes () of the polymerases

 

Hydrodynamics and SAXS Calculations—The programs HYDROPRO (version 5.a) (9) and CRYSOL (version 2.3) (10) were used to calculate hydrodynamic (s20,w) and scattering properties (Rg) from the atomic coordinates in the Protein Data Bank (PDB) files of Taq (1TAQ [PDB] = elongated structure (5); 1CMW [PDB] = compact structure (8)), Klentaq (1KTQ [PDB] ) (19), and Klenow (1KFD [PDB] ) (20). For HYDROPRO, the PDB file, molecular weight, solution density and viscosity, protein partial specific volume, temperature, atomic element radius (AER), and {sigma} values are the HYDROPRO input parameters (9). Hydration of the protein is one of the components contributing to the AER parameter in HYDROPRO. Atomic coordinates for all heteroatoms were deleted from each PDB coordinate file. To ensure that calculated differences between the two conformations of Taq polymerase were not due to different numbers of atoms missing in the two different structures, files were also generated that contained equal numbers of atoms: atomic coordinates missing from 1TAQ [PDB] were deleted from 1CMW [PDB] and vice versa. These deletions did not significantly alter the calculated values. Hydrodynamic and other solution properties were computed for each structure file using an AER value of 3 to construct the primary hydrodynamic particle and six {sigma} values in a range selected to vary the number of beads in the model from ~200 to ~2000. These values are in the parameter ranges suggested by Garcia de la Torre et al. (9).

CRYSOL calculations were performed as described by Svergun et al. (10) on 1TAQ [PDB] (5), 1CMW [PDB] (8), 1KTQ [PDB] (19), and 1KFD [PDB] (20). Program default values were used for all parameters except the scattering q maximum, which was set to coincide with the q maximum of the experimental data sets. The program calculates the theoretical scattering curve from crystal structure coordinates and then alters the volume and hydration contrast parameters to obtain the best fit to the experimental scattering curve. Several crystal structures for Klenow polymerase exist in various functional states. Conformational variations among the different states for Klenow are not significant enough to be easily detectable by SAXS, and CRYSOL calculations with different Klenow structures yield equivalent results (data not shown). Rigid body rotations of the position of the 5' nuclease domain of Taq relative to the Klentaq domain were performed using the program Insight II (Accelrys, Inc.).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Sedimentation Coefficients—Sedimentation coefficients were measured for all four polymerases: Taq, Klentaq, E. coli Pol 1, and Klenow. Measurements were made at 20 °C and were corrected to s20,w values using measured solvent densities and viscosities. Fig. 2 shows representative sedimentation velocity data for Taq polymerase. Values of s20,w were also calculated from crystal structures using the program HYDROPRO from the laboratory of Garcia de la Torre (9). This program uses shell modeling algorithms to predict hydrodynamic properties directly from a PDB file. The measured and calculated s20,w values for the polymerases are reported in Table I.



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FIG. 2.
Representative velocity sedimentation data (upper panel) and equilibrium sedimentation data (lower panel) for full-length Taq DNA polymerase. The velocity sedimentation data show the experimental data along with the fits to the boundaries using the program Svedberg (14, 15). For visual clarity, only a subset of the full set of boundaries is shown, and only half the data points in any single boundary are shown. The lower plot shows the equilibrium sedimentation profile for Taq along with a single species fit. The Residuals plot shown is for the single species fit to the equilibrium sedimentation data. Similar experiments were performed for Klentaq, E. coli Pol 1, and Klenow polymerases, as described in the text. Results are reported in Table I and in the text.

 

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TABLE I
Hydrodynamic properties of the polymerases

 

The two different crystal structures of full-length Taq yield two different calculated s20,w values, as might be expected. The more compact form of the protein would sediment faster than the more elongated form, and its calculated s20,w is thus larger than that of the elongated form. Fig. 3A graphically illustrates the relationships between the measured and calculated data. The measured and calculated s20,w values agree well for Klentaq and Klenow polymerases and for the elongated form of full-length Taq. The measured s20,w value for full-length Taq deviates from the s20,w calculated for the elongated crystal structure by 1.6% and deviates from the s20,w calculated for the compact crystal structure by 5.1%. This is a small difference, but it is well outside experimental error, and its significance can be illustrated by noting that the classic T to R conformational transition for aspartate transcarbamylase (ATCase) is associated with a 3.6% change in its measured s20,w value (21). The deviations between the measured and calculated s20,w values for Klentaq and Klenow are 1.7 and 1.1%, respectively. It is also notable that the measured s20,w value for full-length E. coli Pol 1 is also smaller than the calculated s20,w for the compact conformation of Taq. Since E. coli Pol 1 is ~9 kDa larger than Taq, this further supports the interpretation that Taq is in the elongated conformation. Further, it argues that E. coli Pol 1 is also not in a compact conformation.



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FIG. 3.
Plots of sedimentation coefficients (s20,w values) versus molecular weight for each of the four polymerases. A shows s20,w values plotted directly against protein molecular sizes. Open circles show the experimental values for each of the polymerases. Molecular weights of the polymerases are 62,400 for Klentaq, 68,100 for Klenow, 93,900 for Taq, and 103,100 for Pol 1. The line connecting the experimental data points is only for visual clarity. The open triangles show the s20,w values calculated from the crystal structures using the program HYDROPRO (9) for Klentaq (1KTQ [PDB] (19)), Klenow (1KFD [PDB] (20)), and elongated Taq (1TAQ [PDB] (5)). The open diamond shows the calculated s20,w for the compact Taq crystal structure (1CMW [PDB] (8)). Also shown, for comparative purposes, are the calculated s20,w values for spherical particles of the same molecular weight and partial specific volumes as the polymerases (open squares). B shows an alternate perspective on the hydrodynamic data. Here the experimental values of s20,w for each of the polymerases are shown along with representative data for several other globular and not so globular (e.g. fibrinogen) proteins. The upper solid line shows the (s20,w1/3)/(1-{rho}) versus molecular weight dependence for perfect spheres, whereas the lower line shows this dependence for a set of globular proteins.

 

Fig. 3A also shows the predicted s20,w values for spherical particles of the same molecular weight and for each of the polymerases. These values are not precisely linear due to the slight differences in among each of the polymerases (see below), but they do illustrate, for comparative purposes, the increase in s20,w expected for particles that are increasing in mass but not changing shape. Comparison of the measured s20,w values with these spherical values shows that the full-length polymerases deviate from the spherical limit more so than the Klenow and Klentaq polymerases. Fig. 3B illustrates this same point in a different way. It can be shown by rearrangement of the Svedberg equation that the s20,w values for anhydrous spherical particles are proportional to the 2/3 power of their molecular weight (22). The s20,w values for real globular proteins follow a similar proportionality relationship versus molecular weight (22). Here both full-length polymerases are seen to deviate further from the mean dependence for globular proteins than do their large fragment counterparts, indicating that they are more elongated than their large fragment counterparts.

Effective Stokes radii (RS) for the polymerases can be calculated from the measured s20,w values using the equation: RS = MW(1 {rho})/6N{pi}{eta}s20,w, where MW is the molecular weight, is the partial specific volume, {rho} is the solvent density, N is Avogadro's number, and {eta} is the solvent viscosity (23). Such calculated Stokes radii report the effective spherical radius of a particle having a particular measured s20,w value. They are instructive in reflecting, in angstroms, the relative hydrodynamic sizes of the proteins. The diffusion coefficient (D20,w) for each polymerase can also be calculated from the experimental s20,w and the molecular weight using the Svedberg equation: D20,w = s20,wRT/MW(1 – {rho}w), where R is the gas constant, T is the temperature in Kelvin, MW is the molecular weight of the polymerase predicted from the amino acid sequence, is the partial specific volume of the polymerase, and {rho}w is the density of water. These values are reported in Table I along with RS and D20,w values calculated from the crystal structures using HYDROPRO.

Equilibrium Sedimentation and Partial Specific Volumes—To ensure that the polymerases were monomeric and of high purity and to directly measure the partial specific volumes of each of the polymerases, equilibrium sedimentation experiments were performed. Equilibrium sedimentation runs for all the polymerases fit well to a single species (representative data for Taq polymerase are shown in Fig. 2). Molecular weights for the polymerases measured by equilibrium sedimentation deviated from their known molecular weights by 1% for Klentaq, 2.6% for Taq, 6.3% for Klenow, and 7.5% for E. coli Pol 1. In all cases, the equilibrium sedimentation determined molecular weights were slightly lower than known molecular weights, further indicating the absence of higher order oligomers.

Equilibrium sedimentation was also used to directly measure the partial specific volumes () for each of the polymerases. Values of for the four proteins were both calculated from the primary structure and were measured directly using differential equilibrium sedimentation in H2O versus D2O (16). Fig. 4 shows representative data from the H2O/D2O experiments, and values are shown in Table II. Calculated values have been found to be quite adequate for use in most ultracentrifugation studies, and it is becoming increasingly rare to measure them directly. Small changes in the do not propagate into significant effects on the values of the measured s20,w values. Small changes in the do, however, have quite large effects on the calculated s20,w values estimated using the program HYDROPRO, and this is the primary reason we empirically verified the computational values. It can be seen in Table II that the calculated and measured values are quite similar. The empirical determinations of the values verify that there are no unusual properties of these thermophilic proteins that might skew the calculation of their values relative to the non-thermophilic polymerases.



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FIG. 4.
D2O/H2O sedimentation equilibrium experiments conducted for each of the polymerases. For each plot, the open ovals are equilibrium sedimentation runs in D2O, and crosses are equilibrium sedimentation runs in H2O. Details are described in "Experimental Procedures." Partial specific volumes are determined from the ratio of the D2O/H2O slopes using the method of Edelstein and Schachman (16). Results are reported in Table II.

 

The H2O/D2O determinations themselves are not error-free, of course, and contain small error contributions propagated from the solution density determinations, from completeness of the H2O-D2O exchange, et cetera. Therefore, and because the calculated and measured values are already similar, we have used the average of the calculated and measured values (Table II) in all calculations requiring a value for .

It is notable that the similarity between the values for the mesophilic and thermophilic polymerases suggests that there are no significant differences in the relative packing densities of the different polymerases. This finding is consistent with a recent computational study that compared values calculated from the crystal structures of a large set of mesophilic and thermophilic proteins and found no differences in relative packing densities (24).

Small Angle X-ray Scattering—SAXS experiments on each of the polymerases were performed using synchrotron radiation at several different protein concentrations under the same solution conditions as used to perform the hydrodynamics experiments described above. Guinier plots for each of the polymerases are shown in Fig. 5. Fits of the full scattering curves using the program GNOM (18) are shown in Fig. 6. The measured values for the radius of gyration (Rg) for each polymerase are listed in Table III along with the Rg values calculated from the different crystal structures. Both the program HYDROPRO (9) and the program CRYSOL (10) were used to calculate Rg values from the crystal structure data. Unlike the data for the other polymerases, Guinier plots for E. coli Pol 1 consistently exhibited significant curvature throughout their entire q range at a variety of different protein concentrations and at all SAXS experimental stations used during this study. An example of this curvature is shown in the Guinier plot in Fig. 5. Because of the curvature, E. coli Pol 1 scattering data were not analyzed for Rg values either by Guinier analysis or by GNOM analysis.


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TABLE III
Measured and calculated radii of gyration for the polymerases

 

The SAXS experiments shown also provide evidence that Taq is in an elongated conformation. As seen from the data in Table III, the measured Rg value for full-length Taq agrees most closely with the Rg value calculated from the elongated structure. It should be noted, however, that the Rg data for full-length Taq, like the hydrodynamic data, actually suggests that the solution conformation is in between the two crystal structure conformations. This can be seen by noting that the measured Rg values for Klentaq and Klenow are both larger than the Rg values calculated from their crystal structures. This is a commonly observed discrepancy that is generally attributed to hydration effects (25). This means that if the values could be precisely adjusted for hydration effects, the measured Rg for Taq would likely be in between the Rg values calculated for the two crystal structures. However, the measured Rg would still be much closer to that predicted for the elongated structure. The calculated values do include standard hydration levels of 0.3–0.4 g of H2O/g of protein (and similar hydration levels (~0.4 g/g) are predicted by calculations based on amino acid composition using the program SEDNTERP). However, adjustment of the calculated Rg values to precisely account for hydration is still an active area of research, and as such, no unequivocal guidelines yet exist.

Fig. 7 shows fits of the experimental scattering curves for full-length Taq, Klentaq, and Klenow polymerases overlaid with predicted scattering curves computationally generated from their crystal structures. These fits, generated with the program CRYSOL, begin with a curve generated directly from the known crystal structures and then vary two parameters of the structure-based simulation, the volume and the hydration contrast, to obtain the best correlation between the experimental curve and that predicted from the crystal structure (10). The goodness of the fits for Klentaq, Klenow, and the elongated conformation of Taq are quite similar. Both the statistics ({chi}2) and the visual inspection of the fits in Fig. 7 provide some of the most compelling evidence that Taq is best described as being in an elongated conformation.

The effects of simplistic, rigid body rotations of the position of the 5' nuclease domain of Taq were also examined. Inspection of the two different crystal structure positions of the 5' nuclease domain shown in Fig. 1 shows them to be oriented ~180° relative to each other. Movement of the 5' nuclease domain up to 45° away from its elongated orientation in the 1TAQ [PDB] structure results in relatively small changes in the goodness of fit to the experimental data using CRYSOL. Both slight improvements and slight decreases in the goodness of fit were observed in this range of movement. However, movement of the 5' nuclease domain more than 90° away from its elongated orientation in the 1TAQ [PDB] structure in any direction consistently resulted in significant decreases in the goodness of fit between the experimental and structure-based scattering profiles using CRYSOL. Possible alternate elongated Taq structures are not shown because the precision of the SAXS data at high q values does not allow for further distinction among the possible alternate elongated conformations, even if a more extensive three-dimensional positional grid search were performed. This preliminary modeling exercise does, however, further reinforce the conclusion that the solution structure of Taq is more like the elongated crystal conformation than the compact conformation.

SAXS measurements of the Rg for full-length Taq polymerase were also carried out in the presence of ddATP and in the presence of a matched template primer known to bind stoichiometrically to Taq under the conditions examined (12). The potential effect of ddATP was examined because at least one ddNTP was present at high concentration in the crystallization conditions for 1CMW [PDB] , the compact conformation of Taq. Neither the dideoxynucleotide nor the matched DNA altered the measured Rg. Direct DNA binding studies of Taq to this same DNA also indicate that the 5' nuclease domain is not involved in binding to matched DNA (12). Recent studies from Dahlberg and colleagues (26, 27) have shown that 5' nuclease enzymatic activity of Taq is exhibited on specifically structured bifurcated gapped DNA duplexes. It is not yet known whether both the polymerase and the 5' nuclease domains of a single polymerase molecule can simultaneously act on or bind to one of these more complex duplex structures.

Concluding Discussion—In this study, we have determined some of the basic hydrodynamic and small angle x-ray scattering properties of full-length Taq and E. coli Pol 1 DNA polymerases and their Klentaq and Klenow large fragments. The measured s20,w values, partial specific volumes, RS values, D20,w values, and Rg values have all been determined using established biophysical methods that yield relatively straightforward answers, adding new particulars to the body of data on these important enzymes. What is more equivocal, however, is answering the question: what is the conformation of Taq polymerase in solution? For example, as noted by Svergun et al. (25), direct comparisons between high resolution crystal structures and the low resolution information provided by SAXS must always be viewed with caution, and that even seemingly perfect agreement between the two methods would not be unequivocal proof of the equivalence of the crystal structure and the solution structure. Furthermore, the interrelated fields of calculating hydrodynamic (9) and x-ray scattering properties (10) from crystal structure data are both relatively new, and both still contain clear computational gaps that must be bridged between calculation and experiment (for example, precisely accounting for protein hydration) (9, 10). With these and other caveats in mind, in this study, we have found that every measured hydrodynamic and x-ray scattering property for Taq polymerase consistently correlates with a more elongated conformation of the molecule. Further, when SAXS curves are simulated directly from the crystal structures and fit to the experimental data, the agreement between the experimental data and the elongated structure is clearly better. However, the exact/detailed elongated conformation of Taq in solution is certainly different from the exact elongated conformation in the crystal structure. The data here certainly indicate that Taq in solution is much more similar to the elongated crystal structure than the compact crystal structure, but the hydrodynamic and scattering data presented here do not provide enough structural detail to determine the detailed differences and similarities between the elongated crystal and solution structures. A central goal of this study was to characterize the conformation of isolated/apo Taq polymerase in solution (i.e. without bound DNA) in comparison with the two contrasting crystal structures available for isolated/apo Taq polymerase. It is certainly very possible that, when bound to certain DNA, i.e. during nick translation, the polymerase could move to adopt a compact conformation with both the polymerase and the 5' nuclease domains bound to the same DNA. Further studies are required to begin to explore such possibilities.


    FOOTNOTES
 
* This work was funded by National Science Foundation (NSF) Grant MCB 9904680. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by an NSF-IGERT graduate training fellowship 998703. Back

§ Partly supported by an NSF Research Experience for Undergraduates (REU) supplement. Present address: Duke University Medical School, Durham, NC 27710. Back

To whom correspondence should be sent. Tel.: 225-578-5233; Fax: 225-578-2597; E-mail: licata{at}lsu.edu.

1 The abbreviations used are: Pol 1, E. coli Type 1 DNA polymerase; SAXS, small angle x-ray scattering; PDB, Protein Data Bank; SSRL, Stanford Synchrotron Radiation Research Laboratory; RS, Stokes radius; Rg, radius of gyration. Back


    ACKNOWLEDGMENTS
 
This study would not have been possible without the extensive and generous help of a number of people. We sincerely thank Tomas Plivelic, Cristiano de Oliveira, Hiro Tsuruta, Paul Russo, Jason Bell, Greg Thompson, and Farheen Khan for technical assistance and/or advice. We especially thank Iris Torriani (at Laboratorio Nacional de Luz Sincrotron (LNLS)), John Pople (at SSRL), and Jack Correia (at the University of Mississippi) for their extensive assistance and encouragement. Portions of this study were carried out at the SSRL, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy (DOE), Office of Basic Energy Sciences. The SSRL Structural Biology Program is supported by the DOE and the National Institutes of Health.



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