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.
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ABSTRACT |
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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Sedimentation Coefficient MeasurementsSedimentation 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
DeterminationThe 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 MeasurementsBuffer 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*, where t
= efflux time, V = viscometer constant (in
mm2s2) at temperature T (as supplied by the
manufacturer), and
= buffer density.
Small Angle X-ray ScatteringSAXS 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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Hydrodynamics and SAXS CalculationsThe 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 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
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.).
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RESULTS AND DISCUSSION |
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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. 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
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
)/6N
s20,w,
where MW is the molecular weight,
is
the partial specific volume,
is the solvent density, N is
Avogadro's number, and
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
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
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 VolumesTo 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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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 ScatteringSAXS 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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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 0.30.4 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 (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 DiscussionIn 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.
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FOOTNOTES |
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Supported by an NSF-IGERT graduate training fellowship 998703.
Partly supported by an NSF Research Experience for Undergraduates (REU)
supplement. Present address: Duke University Medical School, Durham, NC
27710.
¶ 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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