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Molecular Dynamics Simulations of Dinucleoside and Dinucleoside-Drug Crystal Hydrates P. Herzyk

a b

a

, J. M. Goodfellow & S. Neidle

b

a

Department of Crystallography , Birkbeck College , Malet Street, London , IE 7HU , WC , U.K. b

CRC Biomolecular Structure Unit , The Institute of Cancer Research Sutton , Surrey , SM2 5NG , U.K. Published online: 21 May 2012.

To cite this article: P. Herzyk , J. M. Goodfellow & S. Neidle (1991) Molecular Dynamics Simulations of Dinucleoside and Dinucleoside-Drug Crystal Hydrates, Journal of Biomolecular Structure and Dynamics, 9:2, 363-386, DOI: 10.1080/07391102.1991.10507918 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507918

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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 9, Issue Number 2 (1991). "'Adenine Press (1991).

Molecular Dynamics Simulations of Dinucleoside and Dinucleoside-Drug Crystal Hydrates P. Herzyk 1' 2, J.M. Goodfellow1* and S. Neidle2 *

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1

Department of Crystallography Birkbeck College Malet Street London WClE 7HU, U.K.

2

CRC Biomolecular Structure Unit The Institute of Cancer Research Sutton, Surrey SM2 5NG, U.K.

Abstract

Molecular dynamics simulations have been performed on the dinucleoside monophosphates rGpC and dCpG, the latter in its intercalation complex with the acridine drug proflavine. The simulations were performed on the crystal structures, with crystallographically-located solvent molecules. It was found that satisfactory results were best obtained with restraints placed on the movements of the water molecules. Motions of individual atoms have been examined in terms of rms fluctuations and anisotropy and correlation functions. Relative motions of groups (pl}osphates, sugars, bases and proflavine molecules) have been analysed.

Introduction Nucleic acids are conformationally flexible. At the polymer and oligomer level, this is manifest in the existence of a number of stable polymorphs, A, B, C, D, Z and their sub-classes (1-3). At a deeper level, sequence-preference structural features have now been observed in a number of crystal-structure analyses and NMR studies in solution [see, for example; 4-6). Molecular mechanics analyses have generally confirmed these findings in terms of static low-energy domains [for example; 7-9), yet few molecular dynamics simulations have been performed on oligonucleotides or on their drug complexes [10-20); these should provide detailed data that is more relevant to solution conditions, as well as quantitating solvent and counter-ion effects. These effects are themselves major determinants of conformational change in nucleic acids. The present paper develops a methodology for analysing dynamics simulations on nucleic acids in their hydrated crystalline environments and examines the dynamics behaviour of two self-complementary dinucleoside monophosphate duplexes, one of which includes an intercalated drug molecule. These systems may represent the * Authors to whom correspondence should be addressed.

363

Herzyk et a/.

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Table I Net Atomic Charges for the Proflavine Molecule in Units of Electron Charge C9 H9 Cl3, Cl2 Cl, C8 HI, H8 C2,C7 H2,H7 C3,C6 Nl5, Nl6 Hl5A Hl6A Hl5B, Hl6B C4,C5 H4,H5 Cll,Cl4 N10 H10

0.040 0.012 -0.033 -0.034 0.109 -0.273 0.125 0.611 -0.754 0.342 -0.480 0.151 0.333 -0.337 0.307

simplest models for double-helical nucleic acids. Both have been previously determined as crystal structures [21-23], with solvent molecules having been defined in the analyses. Monte Carlo simulations of solvent in the proflavine intercalation complex with d(CpG) and in uncomplexed ribo(GpC) have been reported [24,25]. We present dynamics simulations on the same molecular systems in this paper, within their crystallographic environment and we focus on the effects of these environments on the dynamics of nucleic acid conformational behaviour. This study is thus complementary to one on the d(CpG)-proflavine complex, which emphasises the dynamics of the solvent itself [26].

Methods Molecular dynamics simulations were performed on two crystal hydrates namely that of sodium guanylyl-3', 5' -cytidine nonahydrate (GpC) and deoxycytidylyl-3', 5' -guanosine complexed with proflavine (dCpG/proflavine) using version 3.1 of the AMBER package [27]. The all atom force-field was used together with the TIPS3P model for water (28-30]. The coefficients, r* and E, for the non-bonded interactions of the sodium cations were taken from a molecular dynamics study of aqueous N aCI [31] such that r* = 1.525 A and E = 0.045 kcal mol- 1. The proflavine net atomic charges were calculated fitting electrostatic potentials to the atomic charge model using the same method that was used to calculate net charges for the nucleic acids (32] (Table 1). Figures I and 2 detail the numbering schemes used for proflavine and the dinucleosides. As discrete counterion and water molecules were used in these calculations, the dielectric constant was made equal to unity. A non-bonded cutoff of 9.5 A was used in all the simulations together with updating of the non-bonded pair list every 20 timesteps. The SHAKE algorithm was used in order to restrain all bond distances together with a timestep of 0.002 psec. Simulations were performed on an Alliant FX40/3 at the Institute of Cancer Research, and the Cray XMP at the University of London computer Centre.

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MD Simulation of Dinucleoside Complexes

Figure 1: Numbering scheme for rGpC.

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Herzyk et a/.

Figure 2: Numbering scheme for dCpG-proflavine.

GpC Crystal Hydrate

The system consists of four unit cells each with four asymmetric units so that there are 16 GpC molecules with 16 sodium ions and 144 water molecules (1472 atoms altogether) within a monoclinic box of dimensions 21.46X33.854Xl8.664 A and~= 90.54°. Initially, atoms were placed in the positions determined from X-ray crystallography [21 ). As the positions of the water hydrogen atoms were not known experimentally, they were initially oriented toward neighbouring electronegative atoms and then optimized using 650 cycles of conjugate-gradient minimization keeping all other atoms fixed. The whole system was then optimized over 1000 cycles of conjugate gradient minimization, using the default AMBER convergence criterion of the RMS gradient between successive steps being < 0.1 kcal/mole/A. In our initial simulation ofGpC, no restraints were imposed on any atoms (the G 1 simulation). Initial velocities were taken from a Maxwellian distribution at 300K. The system was weakly coupled to a temperature bath at 300K and equilibrated until no significant drifts in root mean square deviation and total energy were observed. The subsequent 40 psec simulation was used in the analysis using a timestep of 0.002 psec and using data collected every 0.05 psec. dCpG/Projlavine Crystal Hydrate

The simulation of the nucleotide/drug complex comprised two complete unit cells consisting of 16 dCpG molecules, 16 proflavine molecules and 216 water molecules

MD Simulation of Dinucleoside Complexes

367

(2088 atoms altogether) in a rectangular box of dimensions 32.991 X21.995X27.018

A. The initial atomic coordinates for the nucleotide and drug molecules were taken

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from the X-ray crystallographic study [22]. Previous Monte Carlo simulations on this system [24] had indicated that there were 108 water molecules in each unit cell and the initial coordinates for these solvent molecules were taken from this previous study. The solvent positions were then optimized using 2000 cycles of conjugate gradient minimization keeping the nucleotide and drug atoms fixed. The position of all atoms was minimized using a further 10,000 cycles of minimization; convergence was much slower than for the GpC system, and a geometric rather than an energetic criterion of convergence was used. Minimisation was terminated when the RMS differences between the positions of non-hydrogen atoms were less than 0.02 A. In the initial simulation of dCpG/proflavine, no restraints were imposed on any atom (the Cl simulation). The initial velocities were taken from a Maxwellian distribution at 298K. The system was then weakly coupled to a temperature bath of the same temperature and equilibrated until no significant drifts in either total energy or root mean square deviation were observed. The analysis was performed over 30 psec using a timestep of 0.002 psec and saving data at every 0.1 psec. Restrained Simulations

Additional simulations were carried out on both crystal hydrates with geometric restraints imposed on the water oxygen atomic positions with respect to their final energy-minimised positions in order to prevent the water molecules drifting too far away from their crystallographically determined positions (the G2 and C2 simulations for GpC and dCpG/proflavine respectively). The initial velocities were taken from a Maxwellian distribution at 5K. The temperature of the systems was then increased to 300K and 298K over the next 4 psec for GpC and dCpG/proflavine respectively. Mter equilibration, 12 psec simulations were carried out with timesteps of0.002 psec and data collected at every 0.1 psec. Analysis of Dynamics Trajectories

Our analysis has concentrated both on changes in the conformation of the duplex nucleotide and drug molecules during the simulations as well as the motion of atoms or group of atoms. Changes in conformation have been followed by calculation of the time-averaged structure (except when the time-course indicated other than unimodal behaviour) and comparison of this with the crystallographic conformation. We have also analysed the data in terms of internal coordinates such as backbone torsion angles, sugar pucker and hydrogen bonding. The motion of atoms has been studied by calculating the root mean square (rms) 2 y, • < ur A • by f1 uctuatwn > , wh"1ch IS• gtven

368

where

Herzyk eta/.

< ilx 2 >

=

< (X-)2 >

A 2 < u.y > = < (Y-)2 >

< u.Z 2 > = < (Z-)2 > A

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and the angular brackets indicate time averages. Average rms fluctuations can be calculated for groups of atoms, < ~r 2 >'12 where the horizontal bar denotes an average over all atoms within the group. These rms fluctuations give a very simple picture of motion within the molecule. Further insight can be made through the calculation of anisotropy and autocorrelation functions. The former parameter is usually described by the calculation of anisotropy ratios, f3 and f2, such that f3 = A2/C2 and f2 = B2/C2

where A2, B2 and C2 are the three elements of the diagonalized mean square displacement matrix for each atom, ordered such that A2 is the smallest and C2 the largest (33]. These elements correspond to the square of the semiaxes of the more traditional thermal elipsoid which are used to describe atomic motion in crystallography. The autocorrelation function for the atomic displacement vector Cr (t) is given by Cr(t)

=

< Llr(s).Llr(t) >/< Llr(s).Llr(s) >s

where Llr (s) andllr (t) are displacement vectors for a given atom at times and timet respectively. The angular brackets indicate averaging over s. From an analysis of these correlation functions, it is possible to classify atoms as having underdamped or overdamped motion [34] as well as to identify groups of atoms which move in a similar way. We have calculated such autocorrelation functions for each atom and averaged them over all duplexes in the system. Finally, we have analysed the simulations in order to study rms fluctuations of torsion angles, sugar pucker and hydrogen bond lengths as these parameters are important in determining the overall conformation of the duplex.

Results We have examined the data from each of the four simulations (G 1, G2, Cl and C2). As each nucleotide duplex was allowed to follow an independent trajectory, we

have analysed 6400 and 2400 duplex conformations from simulations G 1 and Cl respectively and 960 separate conformations for each of the restrained simulations G2 and C2.

369

MD Simulation of Dinucleoside Complexes 2.000

1.500

Cl

simulalion

Gl

simulalion

RMS CAl 1.000

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0.500

0.000

10

0

30

20

SIMULATION TIME

40

Cpsl

Figure 3: Root mean square deviations of non-hydrogen solute atoms between the instantaneous computed structure and the crystal structures, during the course of the simulations.

A

-7200

simulalion

-7400 -7600

Elo L

-7800 -8000 -8200 10

0

B

TIME

( ps)

TIME

Cps l

20

40

30

50

-10300 -10400 -10500 -10600

10

20

30

40

so

60

70

Figure 4: Total energy variations during the equilibration and simulation; (A)- dCpG/proflavine (C I) simulation, (B)- GpC (Gl) simulation.

Herzyk eta/.

370

In order to approximately determine when the system had equilibrated, we have observed rms deviations of the non-hydrogen atoms (Figure 3) as well as the total energy and its components as a function of the length of the simulation (Figure 4). For the initial simulations G 1 and Cl, equilibration appeared to take a relatively long time (32 psec and 22 psec respectively) due to the relaxation of the solvent network The further simulations with restraints on the water molecules (G2 and C2) equilibrated much faster (i.e. within 8 psec). (/)Analysis of GpC Nona hydrate

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Conformation

The rms deviation between the minimized and the crystallographically determined structure is 0.09 A, indicating that the conformations are very similar. However, after the G 1 simulation, the rms deviation has increased to 0.70 A This overall number gives little information on the nature of the discrepancy and can include contributions due to translation and rotation of the duplexes. Therefore, we have analysed the conformation in terms of internal coordinates which are frequently used to describe nucleotide structures. These are presented in Table II. During the unrestrained simulation, the largest changes in conformation occur in the E, ~ and a backbone torsion angles and the sugar pucker (P2) of cytidine. Further analysis showed that these changes occur predominantly in the second strand of each of the eight duplexes. Thus, the average values of the E, ~ and a torsion angles for each strand (a,b) are 195°,88°,298°,48° and 272°,210° respectively. This change in conformation occurs in all eight duplexes during the first simulation. Table II Torsional Angles, Sugar Pucker Coordinates and Hydrogen Bond Lengths for GpC Nonahydrate Crystal Structure

o(G) E

~

a ~ y

o(C) x(G) x(C) P(G) P2(C) Q(G) Q(C) 06... N4 Nl...N3 N2 ... 02

EXP"

OPTb

Gl'

G2d

83 211 292 285 184 50 77 199 208 7 14 0.4 0.4 2.91 2.95 2.86

90 208 285 287 179 51 81 198 211 3 13 0.4 0.4 2.98 2.97 2.90

80.3 ± 6 141 ± 7 173 ± 7 241 ± 8 169 ± 5 81 ± 8 87 ± 5 191 ± 5 195 ± 5 82 ±10 23 ± 9 0.4 ± 0.1 0.4 ± 0.5 2.83± 0.06 2.92± 0.05 2.92± 0.06

87 ± 6 193 ± 7 298 ± 8 273 ±10 172 ± 6 73 ±10 86 ± 5 196 ± 7 203 ± 6 12 ±11 21 ± 9 0.4 ± 0.1 0.4 ± 0.1 2.79± O.Q7 2.91± 0.05 2.95± 0.08

• EXP - results from experimental crystal structure. b OPT- results after optimization using conjugate gradients. ' G I - results after 40 psec MD simulation. d G2 -results after 12 psec MD simulation with constraints on water oxygen positions.

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MD Simulation of Dinucleoside Complexes 1.000

0.800

0.600

RMS !AI 0.400

.. ·

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·.__ .....···.....

........······ ... _:

.:···

..: .....·........ ··.... ··

·....·

...·· ...

0.200

0.000

CYT

GUA 0

10

20

30

40

ATOM NUMBER Figure 5: Root mean square fluctuations of all GpC (one strand) non-hydrogen atoms obtained in the course of MD simulations as well as from the experimentally determined anisotropic temperature factors. The order of atom is as follows: 05' ,C5' ,C4',0 I ',C 1',N9,C8,N7,C5,C6,06,N l,C2,N2,N3,C4,C3',C2' ,02', 03' ,P,OA,OB,05' ,C5' ,C4' ,01 ',C 1',N 1',C6,C5,C4,N4,N3,C2,02,C3' ,C2',02' ,03' ,. The ranges of atoms belonging to both bases and to the adjacent riboses are marked at the bottom of the plots. The assignments of these atoms to individual groups is: Guanine (atom numbers 6-16); Guanosine ribose (atom numbers 1-5 and 17-19); Phosphate (atom numbers 20-24); Cytidine ribose (atom numbers 25-28 and 37-40); Cytosine (atom numbers 29-26).

In the second simulation (G2) in which the water molecules are restrained to their experimentally determined positions, there is no large change in conformation of the second strand and the average values for the backbone torsion angles are very similar to those found in the X-ray structure. Mter performing 12 psec simulations with restraints, these were removed and the simulation continued for a further few psec. Almost immediately the conformation of the second strand changed to that found at the end of the G I simulation. (b) Dynamics

The rms fluctuations of non-hydrogen atoms are presented in Figure 5 for both G I and G2 simulation as well as those calculated from the experimentally determined temperature factors. The fluctuations in atomic positions obtained from the simulation appear to be approximately half those found experimentally which may be due to the inclusion of static disorder in the X-ray analysis. Comparison of rms fluctuations of atoms within the same group (e.g. same sugar ring or same base) shows that there is little change within each group during the simulation. In contrast, the experimental data show some considerable changes in rms displacement for atoms within for example the guanine base. Neither are there great changes in the rms fluctuation of atoms between different groups (Table III) although phosphate atoms (and atoms at

372

Herzyk et a/. Table III RMS Atomic Fluctuations Averaged Over Atoms Belonging to Different Groups a

G2b

EXPC

0.16 0.16 0.18 0.17 0.15 0.26 0.35

0.14 0.18 0.16 0.18 0.15 0.15 0.02

0.35 0.32 0.33 0.34 0.34 0.34 0.64

G1 Guanined Guanine ribose • Phosphate group r Cytosine ribose • Cytosined Na +ion Water oxygen

G 1 - refers to 40 psec MD simulation without restraints. b G2- refers to 12 psec MD simulation with restraints of water oxygen positions. c EXP -refers to experimental results from X-ray crystallography. d This includes all non-hydrogen base atoms. • This includes five non-hydrogen atoms in the sugar ring and the 02' atom. r This includes the phosphorus atom, four neighbouring oxygen atoms and the cytosine C5' atom.

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a

the 5' and 3' ends) are slightly higher than those of atoms within bases or sugar rings with the most mobile atoms having an rms fluctuation of0.36 A Restraining the solvent molecules to fixed positions has very little effect on the rms atomic fluctuations. The atomic anisotropy ratios, f3, are given in Figure 6 for both G 1 and G2 simulations as well as those values calculated from the X-ray crystallographic analysis. It is 1.000

~----------------------------------------------------~

r3

A N I

Gl

G2

0.600

.;-·......· ··....

s

0

T

R 0 p y R A

!. . ·. .

x- ... oy .··':

...... \ :

·.. ·

0.600

\/

0.400

/'··..._........ .

\

:.. ·~.

..

~

n

f~ f \ :·. : : ·..

·..·.:: ~

~

T I

0

0.200

_ __;.:::C.:....Y..:.T_ _----t.~--..,

GUA

0.000 0

10

20

30

40

ATOM NUMBER Figure 6: The f3 anisotropy ratios (as defined in text) of different GpC non-hydrogen atoms obtained in the course of the MD simulations as well as from the experimentally-determined anisotropic temperature factors. The order of atoms is the same as in Figure 5.

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MD Simulation of Dinucleoside Complexes 1.000

type I type II

0.800

0.600

£lr ( L) Downloaded by [New York University] at 04:25 11 May 2015

0.400

0.200

0.000

-0.200

0

10

5

TIME

15

( p s)

Figure 7: ~r correlation functions for the Nl (guanosine 3) atom (type I), and the C5' (cytosine 4) atom (type II) of GpC, from the G I simulation.

immediately apparent that the atomic motions within the simulations are much more anisotropic than found experimentally. Furthermore, the molecular dynamic anisotropy ratios do not change rapidly from atom to atom but show that atoms within each group have similar values. The base atoms show the most anisotropic motion which may be somehow different from that in the rest of the molecule. The ratios for the G 1 simulation averaged over the base, ribose and phosphate group atoms are equal to 0.09, 0.20 and 0.15 respectively. The atomic displacement correlation functions calculated for all non-hydrogen atoms of the GpC system for the G 1 simulation show unimodal behaviour and very rapid decay followed by one damped oscillation and a further series of small high frequency oscillations (Figure 7). The amplitude of the first oscillation as well as the regularity of the following high frequency motions are distinctive. Apart from the atoms at the end of the strands (whose behaviour may be biased by atomic disorder or by a relative freedom of movement) all atoms show correlation functions which fall between two extreme patterns. These extremes are shown by, for example, atom N1 (guanosine 3)(which we have called type I behaviour) and C5' of strand 2 (which we have called type 2 behaviour).

374

Herzyk et a/. Table IV Torsional Angles, Sugar Pucker Coordinates and Hydrogen Bond Lengths in the dCpG-Proflavine Crystal Hydrate EXP"

o(C)

e ~

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a ~

y o(G) x(C) x(G) P(C) P(G)

q(C) q(G) 06... N4 Nl...N3 N2 ...02

84 79 210 203 291 300 289 287 219 218 46 72 94 149 199 195 251 282 22 17 39 170 0.4 0.4 0.4 0.5 2.93 2.77 2.97 2.88 2.81 2.84

OPTh 80 80 214 207 276 293 302 286 211 219 56 69 87 144 193 194 256 281 14 23 19 153 0.4 0.4 0.3 0.4 2.82 2.94 2.87 2.89 2.85 2.79

CJc

C2e

Cl*d

81 82 ± 6 ± 6 93 97 ± 7 ± 6 240 239 ± 8 ± 8 268 272 ± 9 ± 7 276 275 ± 8 ± 7 257 265 ±10 ± 8 301 301 ± 7 ± 7 303 311 ±10 ± 9 211 214 ±10 ± 9 167 172 ±11 ±10 57 57 ± 8 ± 7 69 69 ± 9 ± 8 88 88 ±10 ± 7 111 127 ±13 ±10 191 195 ± 7 ± 7 195 195 ± 8 ± 8 271 270 ± 9 ± 8 261 275 ±10 ±11 4 ± 8 2 ± 8 - 6 ±12 -13 ±12 1 ±13 3 ±14 134 ±12f 135 ±14 ± 11g - 2 0.4 ± 0.04 0.4 ± 0.04 0.4 ± 0.05 0.4 ± 0.05 0.4 ± 0.06 0.4 ± 0.05 0.4 ± 0.05 0.4 ± 0.05 2.81± 0.11 2.84± 0.11 3.73± 0.18 3.32± 0.19 2.92± 0.08 2.87± 0.07 3.51± 0.13 2.17± 0.14 2.96± 0.11 2.89± 0.10 3.14± 0.13 2.93± 0.13

77 79 210 216 277 288 297 292 208 212 63 68 87 139 199 200 251 283 17 21 10 152 0.4 0.4 0.4 0.4

± 6 ± 6 ± 8 ±10 ± 8 ±10 ± 7 ±11 ± 9 ±11 ± 8 ± 8 ± 7 ± 7 ± 7 ± 8 ± 9 ± 9 ±10 ±11 ±13 ±13 ± ± ± ±

0.05 0.05 0.05 0.05

• EXP - refers to experimental results from X-ray crystallography. b OPT - refers to results after optimisation using conjugate gradients. c Cl - refers to 30 psec MD simulation without restraints. d C 1* - refers to the results of the C 1 simulation restricted to the three duplexes of the less distorted geometry (see text). e C2- refers to the 12 psec MD simulation with restraints of water oxygen positions. rvalues refers to the sugar in the C2' endo conformation. gValue refers to the sugar in the C3' endo conformation.

More detailed analysis shows that all base atoms and all Cl' atoms as well as 01' and C2' of cytosine 2 atoms have a first minima lower than -0.2 which implies a large amplitude first oscillation. By contrast, the backbone atoms from the 5' end through to the CS' of cytosine have a first minima value higher than -0.1. Other atoms show intermediate behaviour. All atoms belonging to the same base have very similar correlation functions but the behaviour of each base is slightly different.

375

MD Simulation of Dinucleoside Complexes

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TableV Distances Between the Proflavine Amino and Phosphate Groups (A)

EXP

OPT

C2

CJ

Proflavine Ia Nl6 ... P Nl6 ...05'

4.43 3.80

4.06 3.54

3.74 3.17

3.75 3.02

Nl5 ... P Nl5 ...0A

5.74 4.49

5.31 4.34

5.54 4.72

5.85 5.04

4.07 4.06

3.87 3.84

3.65 3.91

3.78 3.95

Proflavine 2b Nl6 ... P Nl5 ... P

Proflavine I molecule intercalated between the C.G base-pairs. b Proflavine 2 is in the non-intercalated proflavine molecule.

a

Thus, it seems that bases and C 1' atoms have some difference in their motion compared to the backbone atoms, with that of ribose atoms being intermediate. Further analysis of the fluctuations in backbone torsion angles shows that there are two groups with different flexibilities. 8( cytosine), ~ and 8(guanosine) appear more rigid than~. E, a andy. The xangles are correlated with the 8 torsion angles and are therefore relatively rigid. Sugar ring conformation during the G 1 simulation shows that the rms fluctuation of the phase angle P and the puckering amplitude q, are between 8.9-9.6° and 0.040.05 A respectively. This is equivalent to sugar torsional angle fluctuations from v2 = 3.9° to v4 = 6.8 for the guanosine ribose (G) and from v2 = 4.T to v4 = 5.9° for the cytosine ribose. (ii) Analysis of dCpG/proflavine

(a) Conformation

Comparisons of crystal, optimised, and averaged C 1 and C2 structures in terms of torsional angles and interatomic distances are presented in Tables IV and V. The geometry of the dCpG/proflavine complex deviated from the crystal geometry during the course of optimisation and in both the C 1 and C2 simulations. As can be seen from Table IV the optimised structure has the smallest deviation while the C1 average structure has the highest one in terms of torsional angle rms deviations. The non-hydrogen atom rms deviations of optimised, C2, and C 1 structures with respect to crystal one are equal to 0.30 A., 0.55 A and 1.29 A., respectively. The simulation without restraints (C1), led to the most distorted structure, with the backbone angle E switching from a trans to a gauche-conformation in the second strand of all eight duplexes in the early stages of equilibration. This was associated

376

Herzyk et a/.

with significant changes in~ and ~ angles in all eight duplexes, causing some disruption to the base pair hydrogen bonding between guanosine 2 and cytosine 3. These changes affected all eight duplexes. The three least-affected duplexes (1, 2 and 8) have been analysed separately, and are referred to as the Cl * simulation (Table IV).

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In four duplexes the C2'-endo sugar conformation of guanosine 4 switches to C3'endo either in the course of equilibration or during the simulation. None of these duplexes belongs to the set described by Cl *,so that results are not affected by the guanosine 4 sugar pucker changes. Table IV shows that restraining the water oxygens and slowing down the heating procedure employed in the C2 simulation prevented the dCpG/proflavine duplexes from undergoing other types of changes in their geometry during equilibration. The rms deviation of the average backbone torsional angle from the optimised structure was reduced from 24 o to 5o as a result of the C2 simulation. However, the tendency to disrupt the hydrogen bonding between guanosine 2 and cytosine 3 is still apparent The intercalating proflavine is slightly translated and rotated compared to the crystal structure so that it is deeper inside the major groove especially atthe Nl6 end, which is associated with strand I of dCpG. Table V shows that both the Nl6 ... P and Nl5 ... P distances reduce in the course of optimisation from 4.43 A to 4.06 A and from 5. 74 A to 5.31 Arespectively, which results in a parallel shift of the proflavine molecule inside the major groove. The shortest Nl6...05' distance decreases during optimisation from 3.80 A to 3.54 A In the course of both simulations, however, the Nl6 amino group becomes closer to the phosphate group so thatthe Nl6... P distance decreases to 3.74Aand 3.75 A for the C2 and Cl runs respectively, while the Nl5 amino group drifts slightly away from the phosphate group of the second strand. This in tum increases the Nl5 ...P distance to 5.54 A and 5.85 A for the C2 and C I simulations. This results in a slight rotation of the proflavine molecule so that the Nl5 amino group is projected out of the major groove. In the course of the Cl simulation, an extreme situation occurred where in two duplexes there was a permanent hydrogen bond between atoms Nl6 and 05' with an average length of 2.87(10)A In the six other duplexes, this hydrogen bond was only transient. The non-intercalating proflavine is also slightly translated and rotated, which results in it being projected towards the major groove channel. Table V shows that in the crystal structure, the non-intercalating proflavine molecule is situated symmetrically between two phosphate groups of two neighbouring duplexes, with Nl5 ... P and Nl6 ... P distances of 4.06A and ;J.07A respectively. In the course of optimisation these distances decreased to 3.84A and 3.87A respectively, with a symmetrical shifting of the proflavine molecule towards the major groove channel. Molecular dynamic simulations, however, produced some rotation of this molecule which perturbed its symmetric position. On the average, the Nl6 amino group becomes closer to the phosphate groups of the neighbouring duplex, which results in the Nl6 groups being further into the major groove channel than the Nl5 ones. Indeed, in the Cl simulation for four duplexes the Nl6 amino groups make very close permanent contact with phosphate groups of neighbouring duplexes. In two duplexes these contacts occur temporarily, while in the remaining two theN 15 amino groups rather than Nl6 ones have a permanent, close contact with phosphate groups.

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"TOM NUMBER Figure 8: Root mean square fluctuations of different dCpG/proflavine duplex non-hydrogen atoms obtained in the course of the MD simulations as well as from the experimentally determined isotropic temperature factors. The two strands of dCpG comprise atoms I - 38 and 39 - 76, and the proflavine molecules comprise atoms 77-92 and 93- 108. The order of atoms in one strand of the dCpG molecule is: 05',C5',C4',0l',Cl',Nl,C6,C5,C4,N4,N3,C2,02,C3',C2',03',P,OA,OB,05',C5',C4',0l',Cl',N9,C8, N7, C5, C6,06,N l,C2,N2,N3,C4,C3',C2' ,03 '.The order of atoms in a proflavine molecule is: C7,C8,C 12,C9,Cl3, C l.C2,C3,N 15,C4,C ll,N lO,C 14,C5,C6,Nl6. The ranges of atoms belonging to the bases and to the adjacent riboses as well as to both proflavine molecules are marked at the bottom of the plot.

The hydrogen bond pattern in the guanosine2 ... cytosine3 base pair deviates from the standard Watson-Crick one seen in the dCpG/proflavine crystal structure, in the sense that both bases are counter-rotated in the mean base-pair plane, causing a positive shear. This is present in the optimised, C2 and Cl structures, albeit to different extents. The optimised and C2 structures have the N2 atom of guanosine2 interacting via its proton with 02 (2.80A, 2.96A) as well as with N3 (2.97 A, 2.92A). 06(G) ... N4(C) distances are 2.94A, 3.32A while the Nl(G) ... N3(C) distances are 2.88A, 3.17A, respectively. This makes the Nl(G) ... N4(C) distances relatively close (3.08A, 3.10A) despite the steric problems of protons attached to Nl and N4 atoms. In the Cl structure because of the shift mentioned above, a completely new hydrogen bond pattern was found: there is only one strong hydrogen bond, N2(G) ... N3(C) (2.93A). (b) dCpG/Projlavine Dynamics

The rms fluctuations of non-hydrogen atoms for the Cl, Cl *and C2 simulations are presented in Figure 8. For comparison the rms fluctuations calculated from the Xray isotropic temperature factors are also shown. The rms fluctuations averaged over the three least distorted duplexes in the Cl simulation (C 1*)closely match the fluctuations averaged over the whole set of eight

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Figure 9: f2 and f3 anisotropy ratios (as defined in the text) of different dCpG/proflavine duplex nonhydrogen atoms obtained in the couse of the Cl simulation. The order of atoms is the same as in Figure 9.

duplexes, except for the guanine base and ribose of strand 2. These have the largest disorder observed in course of the Cl simulation. Indeed in four duplexes out of the eight the strand 2 guanine ribose changes its conformation from C2'-endo to C3'endo either in the course of equilibration or during the simulation. This accounts for the discrepancy between the Cl and Cl *plots for ribose related atoms i.e. C5',C4', Ol',Cl',C2',C3',N9,C8,N7 (atoms 59-65 and 74-75). The remaining guanine atoms (between 59 and 75) are also affected by the ribose disorder. The C2 simulation atomic rms fluctuations are on the whole smaller than those calculated in the C 1 and C 1* simulations. The correlation between the experimental structure and the C2 simulated one is good, except in the regions of the phosphate groups (atoms 16-21 and 54-59}, where the C2 simulation rms fluctuations are very low. The atoms that were highly mobile in the C 1 simulation (59-75) have lower rms fluctuations in the C2 simulation results. All three simulation profiles in Figure 8 show particularly good agreement for the intercalating proflavine (atoms 77 -92). A characteristic is the high rms fluctuation of the Nl5 atom (85) and a relatively low one for N16 (92). The Nl6 (92). The N16 amino group of the intercalating proflavine is in close contact with the low-mobility phosphate group of strand 1. In contrast, the Nl5 amino group moves outside the major groove, and becomes more mobile. In the case of the non-intercalating proflavine, the three simulations profiles are shifted, but strongly correlated with each other. The significantly lower values for

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Table VI RMS Atomic Fluctuations Averaged over Atoms Belonging to Different Groups

cytosine' Riboser (C) Phosphateg Riboser (G)

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Guanosine' Proflavine 1 Proflavine 2 Water Oxygen

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EXPd

0.31 0.32 0.28 0.27 0.31 0.29 0.31 0.38 0.31 0.38 0.27 0.31 1.22

0.29 0.32 0.27 0.26 0.30 0.27 0.30 0.29 0.31 0.29 0.26 0.35

0.27 0.26 0.21 0.22 0.20 0.22 0.25 0.21 0.27 0.26 0.24 0.26 0.03

0.39 0.45 0.39 0.43 0.47 0.45 0.49 0.34 0.43 0.35 0.56 0.51 0.78

'C1 -refers to the 30 psec MD simulation without restraints. b Cl * -refers to the results of the Cl simulation restricted to the three duplexes with the least distorted geometry (see text). ' C2 - refers to the 12 psec simulation with restraints on solvent atoms. d EXP - refers to experimental results from X-ray crystallography. ' This includes tha all non-hydrogen base atoms. r This includes the five non-hydrogen atoms in the sugar ring. g This includes the phosphorus atom, four neighbouring oxygen atoms and the guanosine C5' atom.

the C2 simulation show that the non-intercalating proflavine is more sensitive to water effects than the intercalated one. The higher values in the Cl *plot maybe due to disorder since one ofthe three C 1* non-intercalatingproflavines is rotated so that its Nl5 amino group interacts with a neighbouring duplex phosphate group while the remaining two proflavines involve N16 amino group interaction. The atomic rms fluctuations obtained from X-ray isotropic temperatures factors tend to be somewhat larger than those calculated. Apart from the disorder region (atoms 59-75) the Cl simulation (Figure 9) shows the highest correlation with experimental results, having a correlation coefficient R of 0.51. The particularly high correlations were found for the non-intercalating proflavine (R = 0.88), as well as for both bases of strand 1 (R=0.66 and R=0.61). The trends in group mobility from the crystallographic data generally show that the phosphate groups are the most mobile and bases are the least, although the differences between them are small. This is apparent from the rms atomic amplitude values obtained from an analysis of isotropic temperature factors (Table VI). This analysis gives average group amplitudes of0.46A, 0.41A and 0.41A for phosphate, ribose and base, respectively. The values from the Cl *simulation are 0.28A, 0.28A and 0.30A, while the ones from the C2 simulation are 0.26A, 0.22A and 0.21A. Figure 9 presents proflles of two anisotropy ratios f3 and t2 for the C 1 simulation. This shows that the atomic motions are highly anisotropic. The f3 ratio profile shows a dependency of the atomic motion anisotropy on the dCpG*/pf subfragments -

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Figure 10: drcorrelation functions for the N3 (cytosine!) atom (class 1), the C3' (cytosine!) atom (class II), and the C9 (proflavine2) atom (class Ia) of the dCpG/proflavine system obtained from the Cl simulation.

minima of this proflle precisely match the bases and the nonintercalating proflavines. Anisotropy values averaged over atoms belonging to different sub fragments are as follows: base- 0.04, ribose- 0.16, phosphate group- 0.08, intercalated proflavine0.13, non-intercalated proflavine- 0.04. The atomic displacement correlation functions calculated for all non-hydrogen atoms of the dCpG-proflavine system can be assigned to one of two classes: I and II. An example is shown in Figure 11 for the cytosine 1C3' atom. These classes correspond directly to those of Swaminathan et al. [20]. Class I has a simple, approximate exponential decay to zero with relatively small high-frequency oscillations superimposed on it. The N3 atom of cytosine 1 is the most pronounced example of this class. The class II correlation function shows an initial exponential decay similar to class I, followed by large, relatively well-defined oscillations. Figure 10 shows the correlation function of the cytosine! C3' atom as an example of this class. Initial comparison of the dCpG atom correlation functions suggests that the class I atoms correspond to the bases while class II are those of phosphate groups and riboses. Closer investigation, however, shows some discrepancies from this simple

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view, which involve atoms that can be regarded as transition ones between the two classes. In both bases of strand 1 and one base (cytidine) of strand 2, atoms close to the ribose show intermediate behaviour. The cytosine 1 Nl atom shows higher correlation with class II atoms than class I ones, while the C6 and CS atoms are intermediate in behaviour. The cytosine3 Nl atom shows much higher correlation with the Cl and 01 atoms belonging to class II, than with its other base atoms. N9, C8 and C4 atoms of guanosine2 are more correlated with its ribose atoms (class II) than with the remaining atoms of guanosine2. In the case of guanosine4, however, the situation appears to be reversed. All guanine atoms as well as the C1 01 and C2 atoms of neighbouring ribose show class I behaviour, while C4 and C3 are intermediate. Thus, base atoms that can be grouped together on the basis of correlation function similarity are: cytosine! - C4,N4,N3,C2,02; cytosine3 - C6,CS,C4,N4,N3,C2,02; guanosine2- N7,CS,C6,06,Nl,C2,N2,N3; guanosine4- 01 ,Cl',C2 ,N9,C8,N7,C5, C6,06,Nl,C2,N2,N3,C4. 1

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All atoms of the non-intercalated proflavine have correlation functions typical of class I, and are highly correlated with both guanines and both guanines and both cytidines of the dCpG duplex. The intercalated proflavine molecule has a more complex pattern of behaviour. Atoms exposed to the major groove channel (from NlS to CS) show Class I behaviour, highly correlated with the non-intercalated proflavine and the dCpG bases. Nl6, C6 and C7 (atoms close to the Nl6 end) show class II behaviour, highly correlated with class II atoms ofthe dCpG strand 1, although not with the phosphate group atoms of strand 2. The carbon atoms exposed to the minor groove channel (C9,Cl3,Cl2,C8 and C 1) have correlation functions typical of class I but with extremely rapid decay and very small relatively high frequency oscillations superposed on it. Figure 7 shows the correlation function of atom C9 as an example of this class (class Ia). Figure 8 shows a time series ofx,y,z components of ~r displacement for atoms typical of class I and II. The y component, the smallest one in both cases, does not have much effect on correlation function. Both x component time series are very much alike in terms of amplitude and frequency of oscillations and do not seem to have a major effect on the correlation function discrimination. However, the z component of the C3 atom shows a mixture oflow and high frequency oscillations which seems to be responsible for the oscillatory correlation function of atoms belonging to class II, corresponding to underdamped motion. The Z component of the N3 atom does not show low frequency oscillations, which indicates that it is the z component which is the cause of distinction between class I and class II atoms. The correlation coefficients of the C3 and N3 time series are 0.48, 0.11 and 0.33 for x,y,z components respectively. This indicates the existence of a rigid body motion in the XY plane. 1

1

Discussion The major findings that emerge from this study are: 1.

Both for GpC and the dCpG/proflavine complex, the calculated rms fluctuations in atom positions are much less than those derived from the experimental temperature factors. The former structure is the more rigid of the two.

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Figure 11: Time series of the Cartesian components of: (A)- N3 (cytosine!) atom. (B)- C3' (cytosine!) atom, in the course of the C I simulation in the system where the z axis is perpendicular to the base planes and the y axis is perpendicular to the interbase hydrogen bonds.

2.

Atomic motions in both systems are generally very similar within an individual group, (especially in GpC) contrasting with the variability observed in the crystal structures.

3.

There are significant differences between the relative mobilities of base, sugar and phosphate groups, for the non-drug system compared to the drug complex.

4.

Simulations without water molecule restraints leads to distorted structures. Conversely, the imposition of restraints procedures simulated structures that are conformationally close to the crystallographic ones.

5.

The intercalated proflavine molecule in the dCpG/proflavine complex is less mobile than its flanking base pairs or the externally-stacked proflavine, and its anisotropic motion is significantly more restricted than these other groups.

6.

In general, motions in the crystal are much larger than those obtained from MD solution simulations [11].

The significant differences between crystallographic and simulated rms fluctuations are undoubtedly due to a number of factors, of which static and/ or dynamic disorder in

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Figure 11 continued

the crystal structure, resulting in increased thermal vibration, is probably a major one. Deficiencies in the force-field parameters, especially for water molecules, may also play a role. It may be that the van der Waals radius forwateris inappropriate for simulation of crystalline hydrates. Analogous differences between crystallographic and simulated rms fluctuations have been reported for bovine pancreatic trypsin inhibitor [37,38].11 is also well-established that temperature factors obtained from crystallographic refinements tend to absorb experimental errors arising from absorption effects, diffuse scattering and incomplete data sets. The large differences in atomic fluctuations and anisotropies of some neighbouring atoms for the crystallographic analysis, are in contrast to the simulated ones (Figures 5,6,8,9), and thus support this conclusions. The differing patterns of relative group motions for the two systems, is noteworthy. In the case ofGpC, the ribose sugar groups are the most mobile and the bases are the least (for the G2 restrained refinement). This order is in contrast with the mobilities obtained from the experimental results [21,35,36], which indicate that the phosphate group is the most mobile. The present results are, however, consistent with a view of A-form RNA (of which GpC can be considered to be a model) with a stiffbackbone due to the C3'-endo sugar, and bases having mobility in the base-pair planes. Figure 6 shows that the bases have the most anisotropic motion in the system. This is consistent with results obtained from analysis of motion in tRNA [33]. The present results are

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suggestive of maximum backbone flexibility being at the P-05' and C5'-C4' bonds (angles a andy), and that the 5'end ribose is more flexible than the 3'end one.

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There are two reasons that may account for the poor correlation of the C 1 plot in the dCpG/proflavine system with the experimental data for the guanine ribose and base of strand 2. Firstly, an artefact caused by the puckering disorder of guanosine4 contributes significantly to the increased values of RMS displacements of atoms 59-75. This is confirmed by the averaged RMS displacement of the guanine and its ribose in strand 2 being greatly increased in the C 1 simulation, as shown in Table V. On the other hand, experimental results show the opposite behaviour (Table V) causing extremely low values of the rms displacements of these fragments. This behaviour is also seen in the C2 simulation results, where the C2 plot correlates much better with the experimental one for atoms 59-75 than the Cl one. Table V shows that the average rms displacements of the guanine and ribose of strand 2 are the lowest in the C2 simulation. It may be that the exceptional C2'-endo puckering of guanosine4 in the crystal structure is imposed by a combination oflocal packing forces and water environment which imposes larger contraints on the motion of this fragment of the complex. This is generally in accord with the greater flexibility of strand 2. The restricted motion of the intercalated proflavine molecule in the drug complex is not in accord with the experimental thermal motions [36], although it does qualitatively agree with that found in the crystal structure of a daunomycin-deoxyhexanucleotide complex (38). The present analysis thus provides support for the view that intercalation of a drug into DNA significantly decreases its local amplitude of motion [38). One ofthe interesting results from our simulations is the effect (or relative lack of effect) on the mobility of atoms when the solvent molecules are restrained to their experimentally-determined positions. For the GpC crystal system, with only nine water molecules per asymmetric unit, there is only a very slight effect on the rms fluctuations (Figure 6). The most noticeable changes are to the phosphate groups, which is unsuprising given their position relative to other atoms in the dinucleotide. There is a noticeable change to the atom anisotropies, but this would be expected if the solvent runs along specific channels in the crystal, as it does in dCpG/proflavine. There is a greater change to the atomic fluctuations in the dCpG-proflavine crystal structure and again the phosphate groups show the most marked changes. This greater effect is no doubt related to the much larger number of water molecules (nearly three times as many) per asymmetric unit. Thus, in summary the effect of restraining the solvent molecules is relatively slight in terms of the motion of the dinucleotide, but the restrains do produce a markedly more acceptable conformation of the molecule, thus emphasising the importance ofincluding solvent molecules explicitly in simulations of biomolecules and in having good models for solvent-interactions. Although there has been a previous simulation (25] on the aqueous hydration of rGpC, it is difficult to make a direct comparison of it with the present simulation. This is in part because our simulation is in the crystal environment whereas that of Subramanian eta/. [25] is intended to model hydration in aqueous solution. This

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work used Monte Carlo simulations and is thus unable to monitor dynamics of the dinucleotide itself The recent molecular dynamics simulation of the dCpG/proflavine complex [26] provides a dynamical view of the hydrogen-bonded water molecule network, and is thus complementary to this study.

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Acknowledgements We would like to thank the SERC for support from the Molecular Recognition IntiativeunderProjectGrantNo. GR/E/51381 (JMG and SN) and GR/F/8107l,(to JMG and others in the Department of Crystallography, Birkbeck College) and the Cancer Research Campaign (Grant SP1384 to SN). JMG gratefully acknowledges a Wellcome Research Leave Fellowship. We are grateful to Dr. C.A Laughton (Institute of Cancer Research) for valuable discussions. References and Footnotes

1. Saenger, W., Principles of Nucleic Acid Structure, Springer-Verlag, New York (1984). 2. Chandrasekaran, R. and Arnott, S., in Landolt-Bornstein, Group Vll Vol 1b, (Saenger W., Ed.) Springer-Verlag, Berlin (1989). 3. Shakked, Z. and Rabinovich, D., Prog. Biophys. Molec. Bioi. 47, 159 (1986). 4. Dickerson, R.E. and Drew, H.R.,J Mol. Bioi. 149, 761 (1981). 5. Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A, Nature 330,221 (1987). 6. Clark. G.R., Brown, D.G., Sanderson, M.R., Chwalinski, T., Neidle, S., Veal, J.M., Jones, R.L., Wilson, W.O., Zon, G., Garman, E. and Stuart, D.l., Nucleic Acids Res. 18, 5521 (1990). 7. Tilton, R.F., Weiner, P.K. and Kollman, P.A, Biopolymers 22, 696 (1983). 8. Rao, S.H. and Kollman, P.A,J Amer. Chern. Soc. 107, 1611 (1985). 9. Tilton, R.F., Weiner, P.K. and Kollman, P.A, Biopolymers 22, 969 (1983). 10. Levitt, M., Cold Spring Harbor Symposium 47,251 (1982). 11. van Gunsteren, W.F., Berendsen, H.J.C., Geurtsen, R.G. and Zwinderman, H.R.J.,Annals New York Acad. of Sci. 482, 286 (1986). 12. Seibel, G.L., Singh, U.C. and Kollman, P.A, Proc. Nat!. Acad. Sci. USA 82, 6532 (1985). 13. Ravishanker, G., Swaminathan, S., Beveridge, D.L., Lavery, R. and Sklenar, H.,J Biomol. Struct. Dynamics 6, 669 (1989). 14. Rao, S.N. and Kollman, P., Biopolymers 29,517 (1990). 15. Singh, I.C., Weiner, SJ. and Kollman, P., Proc. Natl. Acad. Sci. USA 82,755 (1985). 16. Tidor, B., Irikura, K.K., Brooks, B.R. and Karp1us, M.,J Biomol. Struc. Dynamics 1, 231 (1983). 17. Prabhakaran, M. and Harvey, S.C.,J. Phys. Chern. 89, 5767 (1985). 18. Prabhakaran, M. and Harvey, S.C., Biopolymers 27, 1239 (1988). 19. Rao, S.N. and Kollman, P.A, Proc. Natl. Acad. Sci. USA 84, 5735 (1987). 20. Cieplak. P. Rao, S.N., Grootenhuis, P.DJ. and Kollman, P.A,Biopolymers 29,717 (1990). 21. Rosenberg, J.M., Seeman, N.C., Day, R.O. and Rich A,J Mol. Bioi. 104, 145 (1976). 22. Shieh, H.-S., Berman, H.M., Dabrow, M. and Neidle, S., Nucl. Acids Res. 8, 85 (1980). 23. Neidle, S. Berman, H.M. and Shieh, H.S., Nature 288, 129 ( 1980). 24. Mezei, M. Beveridge, D.L., Berman, H.M., Goodfellow, J.M., Finney, J.L. and Neid1e, S.,J Biomol. Struct. Dynamics 1, 287 (1983). 25. Subramanian, P.S., Pitchumani, S., Beveridge, D.L. and Berman, H.M., Biopolymers 29, 771 (1990). 26. Swaminathan, S., Beveridge, D.L. and Berman, H.M.,J Phys. Chern. 94,4660 (1990). 27. Singh, U.C., Weiner,P.K., Ca1dwell,J.W. and Kollman, P.A,AMBER(UCSF)version 3.1, Department of Pharmaceutical Chemistry, University of California at San Francisco ( 1986). 28. Weiner, SJ., Kollman, P.A, Case, D.A, Singh, U.C., Alagona, G., Profeta, S. Jr. and Weiner, P.,J Am. Chern. Soc. 106, 765 (1984). 29. Weiner, SJ., Kollman, P.A, Nguyen, D.T. and Case, D.A, 1 Comp. Chern. 7, 230 (1986). 30. Jorgensen, W.L., Chandrasekhan, J., Madua, J.D., Impey, R.W. and Klein, M.L.,J Chern. Phys. 70, 926 (1983).

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Heinzinger, K and Vogel, P.C., Z. Naturforsch 31a, 463 (1976). Singh, V.C. and Kollman, P.,J. Comp. Chern. 5, 129 (1984). Prabhakaran, M., Harvey, S.C. and McCammon, J.A, Biopo/ymers 24, 1189 (1985). Harvey, S.C., Prabhakaran, M. and McCammon, J.A, Biopo/ymers 24, 1169 (1985). Holbrook, S.R. and Kim, S.-H.,J. Mol.Biol. 173,361 (1984). Aggarwal, A.K and Neid1e, S., Nucleic Acids Res. 13, 5671 ( 1985). van Gunsteren, W.F. and Karp1us, M., Biochemistry 21, 2259 (1982). van Gunsteren, W.F., Berendesen, H.J.C., Herman, S.J., Hoi, W.G.J. and Postma,J.P.M.,Proc. Nat/. Acad. Sci. USA 80,4315 (1983). 39. Holbrook, S.R., Wang, AH.-J., Rich, A and Kim, S.-H., J. Mol. Bioi. 199, 349 (1988). 40. Tang, P., Juang, C.-L. and Harbison, G.S., Science 249, 70 (1988).

31. 32. 33. 34. 35. 36. 37. 38.

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Date Received: July 6, 1991

Communicated by the Editor David Beveridge

Molecular dynamics simulations of dinucleoside and dinucleoside-drug crystal hydrates.

Molecular dynamics simulations have been performed on the dinucleoside monophosphates rGpC and dCpG, the latter in its intercalation complex with the ...
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