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J. Phys. Chem. B 2010, 114, 9934–9945
Effects of an Unnatural Base Pair Replacement on the Structure and Dynamics of DNA and Neighboring Water and Ions K. E. Furse and S. A. Corcelli* Department of Chemistry and Biochemistry, UniVersity of Notre Dame, Notre Dame, Indiana 46556 ReceiVed: June 22, 2010
Incorporating small molecule probes into biomolecular systems to report on local structure and dynamics is a powerful strategy that underlies a wide variety of experimental techniques, including fluorescence, electron paramagnetic resonance (EPR), and Fo¨rster resonance energy transfer (FRET) measurements. When an unnatural probe is inserted into a protein or DNA, the degree to which the presence of the probe has perturbed the local structure and dynamics it was intended to study is always an important concern. Here, molecular dynamics (MD) simulations are used to systematically study the effect of replacing a DNA base pair with a fluorescent probe, coumarin 102 deoxyriboside, at six unique sites along an A-tract DNA dodecamer. While the overall structure of the DNA oligonucleotide remains intact, replacement of A•T base pairs leads to widespread structural and dynamic perturbations up to four base pairs away from the probe site, including widening of the minor groove and increased DNA flexibility. New DNA conformations, not observed in the native sequence, are sometimes found in the vicinity of the probe and its partner abasic site analog. Strong correlations are demonstrated between DNA surface topology and water mobility. I. Introduction Fluorescent probes are extremely valuable tools to study biomolecular structure and dynamics. Fundamentally, their usefulness results from the sensitivity of absorption and emission wavelengths to the immediate surroundings of the probe. Thus, fluorescent probes can be used to report on the dynamics of their local environment. While there are some naturally occurring fluorescent probes in biomolecular contexts, such as tryptophan residues in proteins, often the probe is artificially inserted in place of a native structure or bound as a ligand, raising the question of whether the presence of the probe has altered the local structure and dynamics the experiment was intended to study. The extent to which this is an issue depends on the nature of the probe and the system under investigation, but it must always be addressed. Structural assessments, such as from circular dichroism, quenching studies, biological activity assays, and molecular modeling are often used to confirm that the original biomolecular structure has not been unduly compromised. Understanding DNA structure and dynamics, including the influence of hydration and ions, is of broad importance not only for biological processes, but also new applications in nanoscale assembly that take advantage of the high specificity of DNA base pairing.1 The repetitive structure of stacked base pairs in duplex DNA creates three distinct environments at its interface: the minor groove, major groove, and sugar-phosphate backbone. The grooves are concave (minor more so than major), charge-neutral, and contain potential hydrogen-bonding partners for aqueous solvent and other molecules. The backbone region is convex in shape, polyanionic, and capable of forming chargereinforced hydrogen-bonds. The existence of three very different environments makes oligonucleotides attractive model systems for understanding the role of different factors, such as charge, * To whom correspondence should be addressed. E-mail: scorcell@ nd.edu.
chemical structure, and surface topology, on biomolecular hydration dynamics. Connecting the results of experiments employing molecular probes to specific microscopic motions can be challenging, especially for complex multicomponent biomolecular systems. Molecular dynamics (MD) simulations can aid in the interpretation of results, and, when paired with appropriate control simulations, they can also be used to evaluate the extent of probe perturbation. Using atomistic MD simulations, we have shown that Hoechst 33258, a minor groove binding probe, primarily reports on the dynamics of DNA and water in the backbone region and outer solvation shells of DNA.2,3 In its bound position, Hoechst 33258 is too distant to report on major groove water, and it physically displaces water from the minor groove. Other probes, such as 2-aminopurine, 2-hydroxy-7-nitrofluorene, or coumarin 102, can be used to replace a base or base pair to become an integral part of the DNA structure, where they are ideally positioned to report on the dynamics of the more confined major and minor groove water molecules.1,4-12 However, these can be nontrivial substitutions. Incorporating coumarin 102 deoxyriboside into DNA means replacing an entire base pair, attaching the coumarin to one strand, and removing the partner base from the opposite strand. This replacement, including the introduction of an abasic site analogue, has implications for the local structure and dynamics of the DNA, as well as the surrounding water and ions. Experimental evidence and molecular modeling suggest coumarin substitution has a relatively minor effect on the DNA structure, although a reduction in biological activity was noted.6 While these observations indicate that there is no gross perturbation caused by the coumarin 102 probe, the reduction in biological activity does suggest differences in DNA structure and dynamics related to the probe. It is important to understand these differences and the degree to which the presence of the probe may perturb the very dynamics it is intended to measure, especially in light of the unexpected results of time-dependent Stokes shift experiments for this probe in DNA, for which Berg and co-workers
10.1021/jp105761b 2010 American Chemical Society Published on Web 07/08/2010
Effects of an Unnatural Base Pair Replacement on DNA found evidence of dynamics across a broad 40 fs to 40 ns time scale.1,4-9 The molecular origins of this extremely slow response are not yet fully understood.13 In the present work, we have used MD simulations to systematically investigate the effect of replacing a DNA base pair with a coumarin 102 fluorescent probe. To facilitate comparison, we have chosen the same dodecamer, d(CGCAAATTTGCG), used in our study of Hoechst 33258 bound to DNA.2 This sequence contains a central A-tract, which is known to have a number of unique characteristics, including a narrow minor groove containing geometrically ordered hydration patterns. A high-resolution X-ray crystal structure of this dodecamer shows four layers of ordered water rising from the floor of the groove.14 The primary and secondary layers of A-tracts are often referred to as a “spine of hydration.”15 We have collected and analyzed eight trajectories, totaling over a microsecond of simulation time, in an attempt to comprehensively describe the structure and dynamics of the dodecamer and its surrounding water and ions, both with its native base sequence, and with a coumarin 102 deoxyriboside probe at seven different positions along the double helix. By comparing these simulations, we can describe the effect of probe substitution on DNA structure and dynamics, water translation and rotation, and ion distribution. Understanding how fluorescent probe molecules perturb structure, dynamics, and hydration has broad implications for experimental design and interpretation. In particular, characterizing the effect of coumarin 102 deoxyriboside on DNA hydration is an essential step toward achieving a deeper understanding of the specific biomolecular motions and interactions that give rise to the unexpected time-dependent Stokes shift results, which will be explored in future work. II. Methods A. Molecular Dynamics Simulations. All MD simulations were performed with AMBER 9.0,16 using the parm99 force field17 with parmbsc0 nucleotide modifications18 and the SPC/E water model.19 The development of molecular mechanics force field parameters for coumarin 102 is described in detail elsewhere.20 To create the systems studied here, a high-resolution (1.5 Å) X-ray crystal structure of the extended A-tract DNA dodecamer d(CGCAAATTTGCG) (Protein Data Bank code 1S2R),14 including crystallographic water molecules, was solvated in a cubic periodic box with a minimum buffer of 10 Å between any DNA atom and the closest box edge. Sodium counterions were added to establish charge neutrality. The resulting 27 415 atom system, consisting of the DNA, 22 Na+, and 8859 water molecules, was energy minimized using steepest decent and conjugate gradient methods to relieve any residual unfavorable steric interactions introduced during the solvation procedure. First, the dodecamer was restrained (500 kcal/mol · Å2) while the water and counterions were subjected to 1000 cycles of minimization. Then, the full system was allowed to relax during an additional 2500 cycles of unrestrained minimization. The dodecamer was restrained (25 kcal/mol · Å2) in a 20 ps constant volume MD simulation (NVT), during which water and the Na+ ions were allowed to move freely and the temperature was raised from 0 to 300 K using a Langevin theromostat.21-23 Next, the system was subjected to 150 ps of constant pressure (NPT) MD to achieve proper density, and the solute restraints were gradually reduced from 25 to 5 kcal/ mol · Å2 in five 20 ps stages. The constraints were then released completely for the final 50 ps. Constant pressure was maintained via weak coupling to a Berendsen piston with a 1 ps time constant.24 The cubic periodic box dimensions were then scaled
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9935 to reflect the average periodic box volume from the final 50 ps of NPT simulation. This was followed by 150 ps of constant volume MD (NVT), after which the velocities from the final snapshot were scaled to a temperature of 300 K and the temperature control was switched off for 600 ps of constant energy (NVE) MD. At this point, the equilibrated dodecamer system was used to create the coumarin simulations by manually replacing a purine base with coumarin, then removing its partner pyrimidine to form an abasic site (Figure 1). The coumarin 2′-deoxy-Criboside was designed to serve as a surrogate for native purine/ pyrimidine base pairs10 and has been utilized in time-dependent Stokes shift experiments, typically replacing a G•C pair.4,5,9,25-27 The coumarin probe was modeled in seven different positions, replacing the six unique base pairs in the palindromic sequence, as well as one duplicate position from the opposite end as a convergence check (Figure 1). The new coumarin systems were energy minimized and equilibrated for 50 ps (NVT), after which the temperature was rescaled to 300 K. All eight trajectories were then equilibrated for an additional 10 ns in the NVE ensemble. All eight production MD trajectories were performed in the NVE ensemble with the dodecamer fully flexible, except for all covalent bonds containing hydrogen, which were fixed at equilibrium lengths using the SHAKE algorithm.28 A 2 fs integration time step was used, and configurations were collected every 100 fs. A particle-mesh Ewald summation method was used to compute long-range electrostatic energy and force corrections,29 with a 9 Å real-space nonbonded cutoff. The data collection phase for each of the eight trajectories was 150 ns, for a total of 1.2 µs of simulation data (12 million snapshots). A 10 ns control simulation of bulk water (1114 SPC/E water molecules) was collected using the same simulation conditions. Our previous simulations of coumarin 102 free in solution were used as a second control.3 B. Analysis Methodology. Hydration Structure and Dynamics. In order to characterize water behavior along the length of the dodecamer, we identified the first hydration shell of each of the 12 base pairs. The cutoff for the first hydration shell was defined as R1 ) f(rw + rs), where rw and rs are the van der Waals radii of the water oxygen atom and the nearest solute atom, respectively, and the coefficient f was chosen as 1.1, a value used previously in hydration studies of proteins.30,31 Water molecules were not assigned uniquely to the first solvation shell of a base pair. If a water molecule fulfilled the first shell definition for two different base pairs, it was assigned to both, creating overlap between the results for the individual base pairs. The first hydration shell of each base pair was further subdivided spatially into three “zones,” the minor groove, major groove, and sugar-phosphate backbone region. Although the distinction between the zones is not as straightforward as first versus second hydration shells, we devised a simple and reasonably effective automated selection procedure. Water nearest to a DNA backbone atom was assigned to the “backbone” region; water nearest to a DNA base atom was assigned to one of the grooves. A water molecule was considered to be in the minor groove if (1) it was in the first hydration shell, (2) the closest DNA atom was in the base, not backbone, and (3) the distance to a chosen DNA minor groove marker atom was smaller than the distance to a major groove marker atom. Preliminary testing indicated more accurate zone assignment if backbone atom O4’ was considered part of the “base.” Due to the concave nature of the minor groove, especially in A-tract DNA, some water molecules that we assign to the first hydration shell of the backbone region
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Figure 1. Left panel: schematic of the dodecamer sequence with the coumarin probe substitution sites studied in this work highlighted in orange. Center panel: MD snapshot of the DNA dodecamer with a solvent accessible surface to highlight the groove structure. The first hydration shell is displayed, with water molecules color-coded according to zone: minor groove (red), major groove (green), and sugar-phosphate backbone (blue). Right panels: close-up views of native C•G (position 03) and A•T (position 06) base pairs, compared to a coumarin probe with partner abasic site analogue (position 03). Polar hydrogen atoms have been included, and oxygen (red) and nitrogen (blue) have been colored to highlight potential hydrogen-bonding sites.
could also be classified as secondary and tertiary layers of the minor groove.14 In this work, “minor groove” water refers exclusively to the primary groove hydration layer, which makes contact with the bases along the groove floor. For each trajectory, the first hydration shell of every base pair (or coumarin and partner abasic site) in the dodecamer was monitored to quantify translational motion. The distance each first shell water molecule travels from its initial position was tracked for 10 ps, after which the first shell was reassessed and tracked for the next 10 ps. In this manner, each trajectory was divided into 15 000 unique segments. The distance measurements were then averaged as 〈d2(t)〉, and the linear range of the mean squared displacement curve (t ) 2 - 10 ps) was fit to extract a characteristic slope. This slope is related to the translational self-diffusion coefficient according to the Einstein relation, D ) 〈d2(t)〉/6t. Relatively smaller slopes indicate inhibition of water translational freedom. To assess the error in mean squared displacement, we calculated 〈d2(t)〉 for five nonoverlapping 30 ns sub-blocks of the full 150 ns trajectory, and we report the standard deviation. A similar protocol was used to quantify the first hydration shell water rotational motion using the orientational correlation function
C2(t) ) 〈P2[uˆ(0) · uˆ(t)]〉
(1)
where P2 is the second Legendre polynomial, and uˆ is a unit vector along the water C2 axis. For each trajectory, the first solvation shell of every base pair was identified at t ) 0, and uˆ was monitored for 100 ps. Average C2(t) curves for each trajectory, representing 1500 unique 100 ps segments, were fit to triexponential functions to extract three characteristic time 3 3 ai exp(-t/τi), where ∑i)1 ai ) 1. These time scales, F(t) ) ∑i)1 scales were then combined with their respective amplitudes to
give an average rotational time, 〈τrot〉 ) ∑3i)1aiτi. Here, relatively longer 〈τrot〉 indicate inhibition of water rotational freedom. Standard deviations in 〈τrot〉 were computed from the five 30 ns subsets of the full trajectories. Ion Distribution. The distribution of ions in the unit cell was assessed relative to DNA. For each frame, the ions were assigned to one of three spatial groups: first shell of DNA, second shell, and “bulk” region. The cutoff for the first shell was defined as R1 ) f(ri + rd), where ri and rd are the van der Waals radii of a Na+ ion and the nearest DNA atom, respectively, and the coefficient f was again set to 1.1. The cutoff for the second shell, R2, was defined to be R1 plus f(2ri), representing the diameter of an additional Na+ ion. The ion distribution was averaged over the trajectory, so that 〈N〉first + 〈N〉second + 〈N〉bulk ) 22, the total number of ions in the system. The first shell was further subdivided into three zones, minor groove, major groove, and backbone, as defined above, so that 〈N〉minor + 〈N〉major + 〈N〉backbone ) 〈N〉first. Unlike the water analysis, ions were associated uniquely with the closest base pair in order to scan the ion distribution along the length of the dodecamer. Since the native sequence is a palindrome, this also served as a test of convergence for the native (no probe) simulation, as the distribution should be roughly symmetric about the center, on average, given sufficient sampling. DNA Structure and Dynamics. Mass-weighted root-meansquare deviation (rmsd) of the DNA atoms for each trajectory was measured relative to their positions in the equilibrated structure used to spawn the coumarin simulations. The rmsd was calculated both globally, by fitting the entire dodecamer, and locally, by fitting just the probe and partner abasic site along with the nearest neighbor base pairs immediately above and below. Atomic positional fluctuation (APF) for each nucleotide was calculated relative to average structures generated for each entire trajectory. The width of the minor groove, a general
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structural indicator, was also monitored across each trajectory. It was defined as the distance between a phosphorus atom on one strand and a phosphorus four base pairs away on the opposite strand: positions i and j + 4, where i and j represent a base pair. Ignoring the terminal base pairs, this analysis provides six minor groove distances for the dodecamer studied here, PA6-PC23, PT7-PG22, PT8-PT21, PT9-PT20, PG10-PT19, and PC11-PA18, which span the central A-tract. The AMBER 9.0 module ptraj was used for the DNA calculations.16 III. Results and Discussion A. Water Dynamics. Using simple measurements of water translation and rotation, we have assembled a comprehensive picture of water motion in three distinct zonessthe minor groove, major groove, and sugar-phosphate backbonesalong the length of a DNA dodecamer. Comparing this picture of native DNA to the same analysis with a coumarin probe in seven different positions reveals the extent to which the probe disrupts local water dynamics. NatiWe DNA. For clarity, the mean squared displacement of first solvation shell water, 〈d2(t)〉, was reduced to a single number, the slope of the curve in the 2-10 ps time range (Figure 2). Before 2 ps, 〈d2(t)〉 is nonlinear, and after 10 ps, a significant number of the monitored water molecules (∼30-40%) have exited the first solvation shell cutoff radius. To provide context for the DNA results, two control systems were analyzed: the first solvation shell of coumarin 102 free in solution, “free C102,” and the first solvation shell of a water molecule in bulk water, “bulk.” The two controls gave mean squared displacement slopes of 1.52 and 1.90 Å2/ps, respectively, which translate via the Einstein relation to diffusion coefficients of 2.5 and 3.2 (10-9 m2/s). The latter is comparable to reference values for bulk SPC/E water, 2.8-3.3 (10-9 m2/s).32 Our controls indicate a modest reduction in translational freedom for water solvating coumarin relative to bulk: 25% reduction in mean squared displacement, along with a nearly 2-fold increase in the fraction of water remaining in the first solvation shell after 10 ps, 47% compared to 25% for bulk water. These differences are clearly visible in two-dimensional histograms of first solvation shell translational motion over time (Figure 2), and the inhibition is even more pronounced for the first solvation shell of a G•C base pair or coumarin nucleotide in position 03 of the dodecamer. At this level of analysissconsidering the entire first solvation shell including water solvating the sugar-phosphate backbonesno significant perturbation due to the probe is detectable for this position. The mean squared displacement slopes for the first solvation shell of each of the 12 base pairs of the native sequence, d(CGCAAATTTGCG) is shown in Figure 3a. Since the dodecamer is a palindrome, that is, base pairs 01 f 06 are identical to base pairs 12 f 07, the symmetry of the overall profile about the center provides evidence of sufficient sampling. Water solvating the terminal base pairs (01 and 12) exhibits a slope of ∼0.9 Å2/ps, which is approximately 1.5-2 times slower than the control simulations of coumarin free in solution and bulk water. At the termini, there is little difference in the translational dynamics of water solvating the grooves versus the backbone; however, distinct, zone-specific behaviors emerge for the interior pairs (02-11). The minor groove water is the most translationally inhibited, with slopes of 〈d2(t)〉 approaching 0.3 Å2/ps, which is three times slower than the termini, and 5-6 times slower than control calculations. These results are in reasonable agreement with the recent work of Jana et al., who found a similar retardation of translational dynamics of
Figure 2. Two-dimensional histograms of first solvation shell translational motion over time. The mean squared displacement, 〈d2(t)〉, is indicated by the white curve. The top two panels show water distributions for the control simulations: the first solvation shell of a water molecule in bulk water, and the first solvation shell of coumarin 102 free in aqueous solution. The bottom panels indicate relative inhibition of translational freedom for the first hydration shell at position 03 in the dodecamer when occupied by either the native C•G base pair or a coumarin probe. Significant perturbations due to the presence of the probe are not evident at this level of analysis.
minor groove water that were ultimately attributed to enhanced tetrahedral ordering of water within the groove.33 The slopes of the minor groove mean squared displacement displays a characteristic u-shaped pattern, with the greatest inhibition of translational motion in the central A-tract. The major groove water is significantly more mobile than minor groove water, and there is little difference in the degree of translational inhibition for all of the nonterminal base pairs. In addition, there is a subtle sequence-dependent pattern to the major groove water translational motion, with slight, but reproducible, increased mobility at both ends of the A-tract. Water solvating the sugar-phosphate backbone is the most mobile, but still exhibits a u-shape, becoming more repressed near the center of the strand. This likely reflects the subset of water solvating the backbone near the minor groove, which can also be classified as a secondary layer of minor groove water. Together, the primary and secondary layers of minor groove water comprise the spine of hydration observed in X-ray structures of A-tract DNA.14,15 A similar picture emerges for water rotational behavior (Figure 3b,c). Orientational correlation functions for the first
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Figure 3. Translational (a) and rotational (b,c) behavior of water molecules in the first solvation shell along the native dodecamer. The first hydration shell is further subdivided into the three zones: minor groove (red), major groove (green), and sugar-phosphate backbone (blue). The shaded regions illustrate the standard deviation among averages for the five 30 ns sub-blocks that comprise the full trajectory. (a) Slope of the mean squared displacement, 〈d2(t)〉, for water solvating each base pair. (b) Average rotational times. (c) Close-up of the 0-15 ps range of average rotational times to highlight major groove and backbone details.
solvation shell of each base pair were fit to a triexponential function, then the three correlation times were weighted by their amplitudes and summed to a single number, the average correlation time 〈τrot〉. We note that, for bulk water, correlation times can be obtained using a single exponential fit; however, we found that a single exponential was insufficient to describe the rotational behavior of water solvating DNA. For bulk water, our triexponential fitting procedure gave 〈τrot〉 of 1.4 ps, which is consistent with reference values for bulk SPC/E water (1.2-1.4 ps) calculated using a combination of explicit integration and a single exponential fit.32 Our second control simulation, the first solvation shell of free C102, gave 〈τrot〉 of 2.1 ps, a 1.5-fold reduction in the rotational time scale relative to bulk. These findings are consistent with magnetic relaxation dispersion (MRD) experiments, which give dynamic (rotational) perturbation factors (DPFs), the ratio of the water rotational constant in the vicinity of a molecular interface to the bulk, of 1.0-2.5 for small organic solutes.34,35
Furse and Corcelli Along the DNA dodecamer, the difference in zone behavior is again minimal for the terminal base pairs, but significant for all of the interior pairs. The increase in the rotational time scales for water in the minor groove is dramatic, with 〈τrot〉 approaching 80 ps, corresponding to a DPF of approximately 57, which is consistent with MRD results on DNA hydration dynamics in the minor groove.36,37 Once again, the greatest perturbation in the water rotational correlation times is near the central two A•T pairs. A water occupancy analysis (data not shown) showed a high-occupancy position at the central AT step, where water simultaneously interacted with carboxyl groups of the thymines from opposing strands. Flanking this central water site are similar high-occupancy sites where water bridges N3 and O2 atoms of A and T bases from opposing strands. These bridging water sites have also been identified in X-ray crystal structures of A-tract DNA.14,15 Water in the major groove is much less perturbed rotationally than water in the minor groove, but slightly more affected relative to the sugar-phosphate backbone. Coumarin 102 in DNA. Having established a baseline picture of water solvating the native DNA sequence in our simulations, we can evaluate the effect of inserting a coumarin probe (Figure 4). In general, the effect of coumarin substitution is to increase water translational and rotational mobility compared to native DNA. Replacing either the terminal or second base pair (01 or 02) with the probe results in relatively minor local disruptions of all three hydration zones, affecting the mobility of water solvating the substituted position and, at most, the immediately neighboring base pair(s). Replacing the third base pair, position 03, or analogous position 10 at the opposite end, has a more pronounced, but still local, effect on the minor groove translational and rotational mobility patterns, appearing as an isolated mobility spike at the substituted position. The major groove experiences a more subtle increase in mobility, while the backbone region is largely unaffected. These results are consistent with the fact that the coumarin substitution modeled in this work reduces the number of groups with hydrogenbonding capability in the major groove and eliminates them in the minor groove, relative to a normal base pair (Figure 1). No functional groups are altered in the backbone upon probe substitution, but the creation of an abasic site causes a local increase in DNA flexibility (see below). The effect on minor groove water mobility is far more dramatic when the probe directly infringes on the A-tract, positions 04-06. In addition to a mobility spike at the substituted position, there is increased translational and rotational freedom up to four base pairs away. For positions 05 and 06, coumarin substitution increases minor groove water mobility across the entire central A-tract (04-09), eliminating the characteristic u-shape. Water occupancy analysis still shows high-occupancy water sites along the floor of the minor groove, but reduces to single hydrogen-bond interactions with one strand instead of the simultaneous hydrogen-bonds to bridge opposing strands, as seen in the native simulation. Coumarin substitutions in the A-tract also cause widespread changes in the backbone water mobility patterns, again reducing the u-shape. As discussed for the translational analysis, these changes most likely reflect increased mobility in the subset of water solvating the backbone near the top of the minor groove. High-occupancy water sites in this secondary layer of minor groove water identified for the native sequence were no longer present. The effects of A-tract substitutions on the major groove water are much more subtle, similar in magnitude and extent to the three G•C substitutions.
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Figure 4. Water translational (left) and rotational (center and right) behavior along a dodecamer with coumarin in seven different positions. The orange bar indicates the location of the coumarin probe for each panel. The results for the native sequence from Figure 3 are shown in gray to facilitate comparison. Coumarin substitution has the most dramatic effect on the minor groove, liberating the constricted water molecules.
B. Ion Distribution. For these MD simulations, we used a simple dilute aqueous environment, adding 22 Na+ counterions to balance the -22 net charge of the dodecamer. Ion motions are the slowest simulation component to converge.38,39 In a study of Na+ motions in a similar MD simulation using the Dickerson-Drew dodecamer, d(CGCGAATTGCG), Beveridge and co-workers found that a minimum of 60 ns of MD was required to achieve reasonable convergence and fully characterize the ion distribution.39 Their results suggested that, when calculated for shorter simulations (10 ns), properties that depended on the fine granularity of the ion distribution were susceptible to sampling artifacts, while DNA properties that
depended on mean field effects of the ion distribution converged much more rapidly (within 5 ns). It is currently unknown whether the role of ions in the time-dependent Stokes shift response, if any, is due to granularity or mean field effects of the ion distribution. We analyzed the distribution of Na+ counterions relative to DNA in our simulations to establish convergence and to characterize the effect of coumarin substitution on the local ion population. NatiWe DNA. The ion distribution converged to a reasonable degree within the first 100 ns of the 150 ns trajectory. Since the two strands are identical, we compared the first solvation shell ion occupancy associated with each of the DNA atoms in
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TABLE 1: Average Distribution of 22 Na+ Ions in Seven Different DNA Simulations native 01 02 03 04 05 06
bulka
second
first
backboneb
major
minor
10.85 (0.14) 11.02 (0.12) 11.00 (0.17) 11.31 (0.33) 11.07 (0.23) 11.21 (0.25) 11.43 (0.26)
8.12 (0.15) 8.12 (0.06) 8.16 (0.14) 7.86 (0.17) 8.12 (0.22) 8.14 (0.13) 8.02 (0.21)
3.02 (0.06) 2.86 (0.10) 2.84 (0.06) 2.82 (0.17) 2.81 (0.06) 2.65 (0.14) 2.55 (0.09)
1.96 (0.08) 1.98 (0.07) 2.03 (0.08) 2.01 (0.16) 1.92 (0.09) 1.82 (0.05) 1.67 (0.05)
0.72 (0.08) 0.68 (0.05) 0.56 (0.08) 0.71 (0.03) 0.67 (0.06) 0.69 (0.12) 0.73 (0.07)
0.34 (0.14) 0.21 (0.07) 0.24 (0.05) 0.11 (0.02) 0.22 (0.04) 0.14 (0.06) 0.15 (0.03)
a Simulation box spatially subdivided by DNA solvation shell: 〈Nion〉bulk + 〈Nion〉second + 〈Nion〉first ) 22. subdivided into backbone, major, and minor groove zones: 〈Nion〉first ) 〈Nion〉backbone + 〈Nion〉major + 〈Nion〉minor.
one strand, A, to its partner strand, B (data not shown). At 30 ns, this analysis revealed an initial bias with greater overall ion density for A (slope of 0.59), and a significant amount of spread (correlation coefficient, r, of 0.72). The results improved steadily over time, to a point of diminishing return around 100 ns. For the full 150 ns, the correlation coefficient was 0.93, and the total ion occupancies for the first solvation shell of the two strands are the same within error, 1.53 ( 0.07 and 1.49 ( 0.05, respectively. If we expand the analysis to include the first two solvation shells, a cutoff distance of around 5.6 Å, then r ) 0.98, comparable to correlations reported for the Dickerson-Drew dodecamer using a 5.0 Å cutoff.39 On average, approximately half of the 22 Na+ ions occupied the areas we defined as the first and second solvation shells, ∼3 and ∼8, respectively, with relatively lower occupancy at the termini (Table 1). Of the three ions in direct contact with DNA, 2 were associated with the backbone, 0.7 were in the major groove, and 0.3 in the minor groove. These fractional occupancies can also be interpreted as having one ion present in the minor groove 30% of the simulation, or two ions 15% of the time. Localization of ions in the minor groove is a known property of A-tracts, shown by both experimental and theoretical studies.39-42 For the first 120 ns of the simulation, there were two high-occupancy ion positions in the minor groove, near the 5′ and 3′ ends of the central A-tract, consistent with previous MD findings for the Dickerson-Drew dodecamer.39 In the final 30 ns, however, an ion entered the minor groove at the central AT step. This region is known to be an electronegative pocket,40 but exhibited very low occupancy in the 60 ns Dickerson-Drew simulation. Significant population of this site late in the trajectory suggests that a complete understanding of the fine details of ion distribution around DNA, especially atypical structures such as A-tracts, may require even longer simulations or enhanced sampling techniques. Coumarin 102 in DNA. Replacing a base pair with coumarin has only a minor effect on the distribution of ions between the first, second, and outer shells of DNA. The scans of ion occupancy in the grooves and backbone region show some qualitative trends, but, not surprisingly, also exhibit the largest errors of any property measured in this work. With an average of 2-3 ions in the first solvation shell of the entire dodecamer, spatially subdividing the ion occupancy into zones surrounding individual bases stretches the limits of statistical significance. In order to get more quantitative results, we summed the ion occupancy in the various shells and zones for all 12 base pairs (Table 1). This reveals a net movement of approximately half an ion on average from the first solvation shell to the bulk solvent as the coumarin substitution nears the center of the dodecamer. This ion density is lost from the minor groove and backbone region; the major groove and second solvation shell show no statistically significant change in average occupancy. This local ion depletion in the minor groove could result from
b
First solvation shell further
electrostatic, structural, or dynamic changes in DNA related to the presence of the probe. C. DNA Structure and Dynamics. Although the structure of double stranded DNA is relatively simple compared to proteins and even RNA, dozens of different geometric quantities are necessary to fully characterize its structure. Monitoring all of these structural indicators is beyond the scope of the present work; however, the Ascona B-DNA consortium (ABC) has comprehensively assessed the effects of base sequence on the structure, dynamics, and interactions in B-DNA.43 Presently, we chose to focus on three of the most general structural indicators: the width of the minor groove, atomic position fluctuations of each individual base, which is analogous to a crystallographic B factor, and rmsd from a reference structure. The trends in these quantities along the dodecamer with and without coumarin reveals the effect of probe substitution on the structure and conformational flexibility of DNA. NatiWe DNA. The bases in each strand of DNA are connected by a negatively charged backbone of alternating pentose sugar rings and phosphate groups. Conformational changes in this sugar-phosphate backbone involve a complex interplay of seven different torsion angles. One simple way to look at the average effect of these motions is to measure the width of the minor groove. To give the shortest distance across the groove, the minor groove width was defined as the distance between a phosphorus atom on one strand and a phosphorus four base pairs away on the opposite strand. This analysis provides six measurements for the dodecamer studied here, describing the width of the central A-tract, positions 04-09 (Figure 5a). For the native DNA simulation, the minor groove width profile is u-shaped, narrowing by nearly 4 Å in the center. The profile is more symmetrical than the minor groove width profile from the X-ray structure,14 and is stable across the simulation. We also measured the APF for each of the 24 nucleotides (base plus backbone), in order to evaluate the flexibility of the two strands independently (Figure 5b). The DNA sequence is palindromic, which means that the two individual strands are identical, 5′-CGCAAATTTGCG-3′. In a double helix, the strands run opposite to one another, or antiparallel, so when plotted as base pairs, the fluctuation profiles for the two strands are related by reflection symmetry about the center. The modest symmetrical profiles with small standard deviations indicate consistent fluctuation about a stable average structure. The exception is terminal base pair 12, which clearly frays during the simulation. The large fluctuation in the cytosine of base pair 12 indicates that it flips out into solution, while the guanine remains stacked with neighboring bases. The effects of this fraying are localsthe fluctuation of neighboring base pairs is not significantly different from their analogues at the opposite end. Interestingly, the profiles demonstrate sequence specific differences. The 5′-CGCAAA half exhibits greater average
Effects of an Unnatural Base Pair Replacement on DNA
Figure 5. Structural and dynamic characterization of the native dodecamer. The shaded regions in panels a and b illustrate the standard deviation among averages for the five 30 ns sub-blocks of the full trajectory. (a) Average minor groove width, 〈dmwg〉, illustrating the characteristic A-tract minor groove narrowing. (b) Average APFs for the 24 individual bases for the two identical antiparallel strands (green and purple). The poly-A stretches show reproducibly larger fluctuations than their partner T bases, and increased fluctuation at position 12 is due to the pyrimidine base, C, opening out into solution. (c) Global mass-weighted rmsd for the atoms of all 12 base pairs, relative to the equilibrated structure used to spawn the probe simulations. The conformational change visible at ∼35 ns is the transient fraying at terminal position 12.
fluctuation than TTTGCG-3′ for both individual strands of the doublehelix,resultingins-shapedfluctuationprofiles.Pyrimidinepurine base pair steps, such as CA, are known for their flexibility, and have been suggested to act as hinges in DNA-protein binding.43 Our results show greater inherent flexibility in the strand containing the CA step, relative to their partners in the complementary strand (G and T), and describe a sequence-specific dynamic signature that can be evaluated in the coumarin simulations. Coumarin 102 in DNA. The minor groove width is largely unaffected by coumarin substitution at the terminal two positions, 01 and 02 (Figure 6). Substitution at the third position (03 and 10) narrows the minor groove at the neighboring base pair, while widening the center of the groove slightly. The former narrowing effect is dramatic for coumarin substitution at position 03, and much more subtle for its analogue at the opposite end of the dodecamer, position 10. In contrast to the modest effect of the G•C substitutions, placing the probe directly
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9941 in the central A-tract destroys the minor groove width pattern seen with the native sequence, partially for position 04 and completely for 05 and 06. Clearly, the substitution alters something fundamental to A-tracts. Both intrinsic structural and electrostatic arguments have been proposed to explain structural features of DNA, such as groove width.44 Since the coumarin substitution alters both base-base interactions and the electrostatic environment, directly through the reduced polarity relative to a native base and indirectly through the local ion depletion described above, our results do not argue more strongly for either. Coumarin substitution also has a noticeable effect on the APF patterns along the dodecamer (Figure 6). Not surprisingly, all of the coumarin simulations show a local increase in flexibility for the abasic site analogue, where the sugar ring is no longer tethered to a base. The extent of perturbation beyond the abasic site, itself, depends on sequence and relative position in the dodecamer. The perturbation is much more widespread for coumarin substitutions in the central A-tract, and include changes in the behavior of the coumarin-containing strand, as well as the abasic site strand. NMR experiments have shown that abasic sites, which are common DNA lesions, can adopt both intrahelical and extrahelical configurations and perturb local conformation, generally at and directly adjacent to the site of damage.45 Other NMR studies have suggested more widespread structural changes, up to four base pairs away, for abasic sites in A-tract DNA, compared to primarily nearest neighbor effects in mixed sequences.46 The results in Figure 6 also suggest widespread, nontrivial changes in the flexibility and dynamics of A-tract DNA when a base pair is replaced by coumarin and an abasic site analogue. The first A-tract position, 04, yields particularly interesting results. The increases in fluctuation are relatively widespread, and include a spike in flexibility at the abasic site and a broader hump in the coumarin-containing strand near base pairs 05 and 06. A plot of local rmsd versus time, calculated for the coumarin site and immediately neighboring base pairs, suggests discrete conformational changes near coumarin across the entire the simulation (Figure 6, first column). DNA conformations sampled from regions of interest on the rmsd plot are shown in Figure 7a. The changes in rmsd relate to reversible flipping of the abasic site analogue, from an intrahelical position in conformations 2, 4, 6, 8, and 10, to an alternate extrahelical position in 1, 3, 5, 7, and 9. In the extrahelical conformation, coumarin shifts toward the abasic site analogue, and a bulge appears in the backbone of the coumarin-containing strand between base pairs 05 and 06. The two conformations interchange reproducibly throughout the trajectory, explaining the features noted above for the APF pattern. Surprisingly, a similar analysis of the native simulation with an A•T base pair in position 04 reveals transient adoption of a conformation similar to the alternate, extrahelical structure seen with coumarin, including the bulge between base pairs 05 and 06 (Figure 7b). It is a statistically rare event, happening once in the 150 ns simulation. These results suggest that the introduction of coumarin and the abasic site analogue may stabilize an energetically unfavorable conformation of native DNA, altering the conformational dynamics of this sequence. A similar conformational analysis of coumarin substitution in positions 05 and 06 demonstrates more rapid interconversion between intrahelical and extrahelical sugar positions, along with a range of intermediate structures (Figure 8). The backbone bulge in the coumarin-containing strand, noted for
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Figure 6. DNA behavior with coumarin in the seven different positions shown in Figure 1. (left) Local rmsd calculated for the probe substitution site and immediate neighbor base pairs (blue). For comparison, the gray curves show analogous results for the two equivalent positions in the native simulation. (center and right) Average minor groove width, 〈dmwg〉, and APF. The orange bar indicates the location of the coumarin probe, and results for the native sequence from Figure 6 are shown in gray for comparison.
position 04, is still visible but much more subtle for positions 05 and 06, suggesting some sequence dependence for the feature. The terminal substitution, position 01, is surprisingly stable. The untethered terminal abasic site analogue fluctuates wildly, but the flexibility of the strand containing coumarin is indistinguishable from the native strand, indicating that the probe remained stacked with the double helix. Substitution at the third position gives slightly different results for analogous positions 03 and 10, with the latter exhibiting increases in both the average fluctuation and standard deviation for the coumarin-containing
half of the dodecamer, as well as more prominent changes in local rmsd. Further analysis demonstrates that the two simulations actually sample similar conformations, representing both intra- and extrahelical abasic site analogues, but with different interconversion rates (Figure 8). Position 10 also exhibits a large increase in rmsd at the end of the trajectory, which reflects a far more significant conformational change in which the abasic site is extrahelical, the cytosine in neighboring base pair 11 flips out into solution, coumarin stacks with the abandoned guanine, and the terminal base pair, 12, loses Watson-Crick pairing. The extent of perturbation is likely an end effect, facilitated by
Effects of an Unnatural Base Pair Replacement on DNA
Figure 7. DNA conformations sampled from MD simulations. (a) Coumarin nucleotide in position 04. Conformations are average structures for 100 ps segments of the trajectory taken from the indicated regions on the plot of local rmsd versus time. The even numbered conformations (blue) have low rmsd, and represent structures with an intrahelical abasic site analogue. The odd numbered conformations (green) have relatively high rmsd, and all belong to a cluster of structures with an extrahelical abasic site analogue and a reproducible bulge in the backbone of the opposite strand between base pairs 05 and 06. (b) Conformations for the native DNA sequence with an A•T pair in position 04, showing a rare occurrence of a conformation, 5, similar to the alternate structure seen with coumarin.
the proximity of the probe to the terminus. The variations in the results suggest base pair replacement at this third position is volatile, and capable of destabilizing the entire terminus. The simulation with coumarin in position 02 is anomalous compared to the rest of the set. While the average fluctuation profiles are qualitatively similar, the standard deviations are dramatically larger, indicating significant conformational diversity during the trajectory. Analyses of APFs for the five 30 ns sub-blocks, along with rmsd versus time (Figure 6, first column), show a conformational change near the probe around 100 ns, and base pair fraying at the opposite end in the final 30 ns. While we cannot rule out a causal relationship between the two events, that conclusion is not supported by the current simulations, which show terminal fraying in the absence of coumarin substitution (native sequence) and local conformational diversity near coumarin without base pair fraying at the opposite end (position 10). These increases in conformational diversity and APF do not have a strong influence on water behavior: while the simulation with coumarin in position 02 is an outlier in the fluctuation analysis, it is nearly indistinguishable from the native profile in the water analyses. For position 02, the first solvation shell, 〈τrot〉 < 10 ps, adjusts much faster than the conformational changes, which occur on a nanosecond time scale.
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9943 Comparing Figures 4 and 6, it is clear that the water mobility profiles for the minor groove mirror the patterns in minor groove width. The relationship between minor groove narrowing and ordered water in sets of X-ray structures was previously observed by Chui and Dickerson.47 The partial groove widening with coumarin in position 04 is correlated to an increase in water mobility in that same region, and the comprehensive groove widening with coumarin at positions 05 and 06 correlates to the widespread loss of the u-shaped water constriction. Even the more subtle features, such as the pattern of minor groove width changes noted for coumarin substitution at position 03/ 10, are reflected in the water profiles: water mobility decreases at the probe’s neighboring base pair where the minor groove narrows, and increases in the center of the dodecamer where the minor groove widens, and the effect is more pronounced for 03 than 10. This relationship between DNA surface topology and water behavior is consistent with the picture of protein hydration dynamics derived from MRD studies.34,37,48 In the MRD picture, surface depressions impose geometric constraints on water molecules, inhibiting the cooperative motions that give rise to the fast rotational and translational dynamics in the bulk. Surface topology is the critical variable, thought to play a larger role in the constriction of water motion than chemical structure, charge, or polarity. In our work, water solvating the concave neutral grooves is more constricted than water solvating the negatively charged, but convex, backbone. The more narrow the groove, the more constricted the water motion. This result is also consistent with ultrafast infrared pump-probe spectroscopy studies in reverse micelles, which demonstrated that confinement, not charge, was the primary factor governing the dynamics of nanoscopic water.49 IV. Conclusions A-tract DNA, which is known for its unusual, sequencespecific structural features, provides a stringent testsin many ways a worst case scenariosfor evaluating the perturbation caused by introducing a fluorescent probe into a biological molecule. Indeed, we find that, while the overall structure of DNA is fairly robust to base pair replacement probe substitution, sequence specific features, such as minor groove narrowing, are far more fragile. Although it may seem obvious that replacing one of the A•T pairs will disrupt sequence specific characteristics because you are literally changing the sequence, the extent of the disruption is still surprising. Unlike a G•C base pair, the coumarin nucleotide is not attached to the opposite strand, and could, conceivably, adapt its position to retain the native A-tract structure. Instead, we see widespread disruption of characteristic A-tract features, including minor groove narrowing, water confinement and localization of monovalent cations. Major groove hydration and ion occupancy, on the other hand, are negligibly affected. The relationship between DNA surface topology and water dynamics, suggested by MRD studies, is clearly demonstrated in the minor groove in this work. On the basis of the atomic fluctuation analysis, the abasic site analogue is at least as disruptive to DNA structure and dynamics as the probe itself, contributing to changes in local dynamics, conformational sampling, and the average structure. The sugar ring can adopt different intra- and extrahelical conformations, allowing the coumarin nucleotide to shift in toward the center of the double helix, which can be accompanied by nontrivial conformational changes in the backbone. While the extent of the conformational changes does appear to have some sequence dependence, the general
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Figure 8. DNA conformations sampled from MD simulations with coumarin replacing the base pair in (a) position 03, (b) position 10, which is analogous to 03, (c) position 05, and (d) position 06. Conformations are average structures for the 100 ps segments indicated on the local rmsd plots. Structures with relatively low rmsd and primarily intrahelical abasic site analogues are colored blue. Structures with relatively higher RMSDs, most of which have more extrahelical abasic site analogues, are colored green. Structures are clustered according to conformational similarity. The structures in A are rotated 180° so that position 03 is at the bottom of the dodecamer to facilitate comparison to 10.
findings should not be unique to A-tracts. Taken together, these results suggest that this probe system does not report on normal DNA dynamics, but rather dynamics near an abasic site analogue, which appears to have a different signature. This could have important implications for the study of DNA damage. The results also have some important implications for experimental design. They clearly demonstrate that this probe cannot be placed directly in an A-tract if the goal is to study highly constrained minor groove water. The directly adjacent G•C position (03 or 10 in this work) is the closest the probe can be inserted without significantly disrupting the water behavior. Even then, probe range and the increased mobility of the water directly solvating the probe, along with potential structural deformations and dynamic changes related to the probe and abasic site, should be taken into account when interpreting the experimental results. Finally, single base
replacement probes that do not require the introduction of an abasic site may be a safer choice to study normal DNA dynamics. Acknowledgment. S.A.C. gratefully acknowledges support from the National Science Foundation (CHE-0845736). In addition, the authors are thankful for support from the Center for Research Computing at the University of Notre Dame. References and Notes (1) Berg, M. A.; Coleman, R. S.; Murphy, C. J. Phys. Chem. Chem. Phys. 2008, 10, 1229. (2) Furse, K. E.; Corcelli, S. A. J. Am. Chem. Soc. 2008, 130, 13103. (3) Furse, K. E.; Lindquist, B. A.; Coreelli, S. A. J. Phys. Chem. B 2008, 112, 3231. (4) Andreatta, D.; Lustres, J. L. P.; Kovalenko, S. A.; Ernsting, N. P.; Murphy, C. J.; Coleman, R. S.; Berg, M. A. J. Am. Chem. Soc. 2005, 127, 7270.
Effects of an Unnatural Base Pair Replacement on DNA (5) Andreatta, D.; Sen, S.; Lustres, J. L. P.; Kovalenko, S. A.; Ernsting, N. P.; Murphy, C. J.; Coleman, R. S.; Berg, M. A. J. Am. Chem. Soc. 2006, 128, 6885. (6) Brauns, E. B.; Madaras, M. L.; Coleman, R. S.; Murphy, C. J.; Berg, M. A. J. Am. Chem. Soc. 1999, 121, 11644. (7) Brauns, E. B.; Madaras, M. L.; Coleman, R. S.; Murphy, C. J.; Berg, M. A. Phys. ReV. Lett. 2002, 88, 158101. (8) Brauns, E. B.; Murphy, C. J.; Berg, M. A. J. Am. Chem. Soc. 1998, 120, 2449. (9) Sen, S.; Andreatta, D.; Ponomarev, S. Y.; Beveridge, D. L.; Berg, M. A. J. Am. Chem. Soc. 2009, 131, 1724. (10) Coleman, R. S.; Madaras, M. L. J. Org. Chem. 1998, 63, 5700. (11) Dallmann, A.; Pfaffe, M.; Mugge, C.; Mahrwald, R.; Kovalenko, S. A.; Ernsting, N. P. J. Phys. Chem. B 2009, 113, 15619. (12) Pal, S. K.; Zhao, L.; Xia, T. B.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13746. (13) Furse, K. E.; Corcelli, S. A. J. Phys. Chem. Lett. 2010, 1, 1813. (14) Woods, K. K.; Maehigashi, T.; Howerton, S. B.; Sines, C. C.; Tannenbaum, S.; Williams, L. D. J. Am. Chem. Soc. 2004, 126, 15330. (15) Drew, H. R.; Dickerson, R. E. J. Mol. Biol. 1981, 151, 535. (16) Case, D. A.; Darden, T. A.; Cheatham, T. E.; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley, M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell, S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.; Kollman, P. A. AMBER 9; University of California, San Francisco: San Francisco, 2006. (17) Wang, J. M.; Cieplak, P.; Kollman, P. A. J. Comput. Chem. 2000, 21, 1049. (18) Perez, A.; Marchan, I.; Svozil, D.; Sponer, J.; Cheatham, T. E.; Laughton, C. A.; Orozco, M. Biophys. J. 2007, 92, 3817. (19) Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. J. Phys. Chem. 1987, 91, 6269. (20) Furse, K. E.; Corcelli, S. A. J. Chem. Theory Comput. 2009, 5, 1959. (21) Izaguirre, J. A.; Catarello, D. P.; Wozniak, J. M.; Skeel, R. D. J. Chem. Phys. 2001, 114, 2090. (22) Loncharich, R. J.; Brooks, B. R.; Pastor, R. W. Biopolymers 1992, 32, 523. (23) Pastor, R. W.; Brooks, B. R.; Szabo, A. Mol. Phys. 1988, 65, 1409. (24) Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684. (25) Sen, S.; Gearheart, L. A.; Rivers, E.; Liu, H.; Coleman, R. S.; Murphy, C. J.; Berg, M. A. J. Phys. Chem. B 2006, 110, 13248.
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