6790
J. Phys. Chem. 1992, 96, 6790-6800
(18) Kroger, F. A.; Vink, H. J.; Volger, J. Resistivity, Hall Effect and Thermo-Electric Power of Conducting and Photo-Conducting Single Crystals of CdS from 20-70 OK. Physica 1954.20, 1095-1096. (19) Neumcke. B.: Bambera. E. The Action of Uncouolers on Lioid Bilayer kelembronesl in ‘Membra&; Eisenman, G.,-Ed.; Marcel Dekkdr, Inc.: New York, 1975; pp 215-249. (20) Trissl, H. W.; Liuger, P. Photoelectric Effects at Lipid Bilayer Membranes: Theoretical Model and Experimental Observations. Biochim.
Eiophys. Acta 1972, 282, 40-50. (21) The energy difference of an ion in two media of dielectric constant c I and c2 is given by the Born’s formula
-[-
I
= z2e2 1 -
2r
e,
J:
\
where r is the ion radius, z is the valency, and e is the elementary charge,
Self-Aggregation of Guanosine 5’-Monophosphate Studied by Dynamic Light Scattering Techniques W. Eimer* and Th. Dorfmiiller Department of Chemistry, University of Bielefeld, 0-4800 Bielefeld 1, FRG (Received: March 16, 1992)
The specific self-aggregation of sodium guanosine 5’-monophosphate (5’-GMP) in aqueous solution at different NaCl concentrations was studied by dynamic light scattering techniques. Depolarized dynamic light scattering (DDLS) monitors the rotational motion of the aggregate structures. Two relaxation processes could be observed as a function of temperature and concentration. The fast mode, in the range of a few hundred picoseconds, is associated with the reorientation of stacked monomers, while the slow process is determined by the motion of specific tetramer stacks, a unique aggregate structure of 5’-GMP. The rotational motion of both species is described by hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre for cylinder-symmetric molecules provides good predictions for the average dimensions of the monomer and tetramer stacks, formed at different concentrations. A comparison with the Perrin equations for biaxial ellipsoids is given. The addition of sodium chloride to the mononucleotide solutions favors the formation of stacked tetramers. The translational motion of 5’-GMP was studied by photon correlation spectroscopy. In accordance with the DDLS results, the intensity autocorrelation function is characterized by one or two relaxation processes, depending on the experimental conditions (concentration, temperature). A combination of the two transport coefficients, in connection with the hydrodynamic model of Tirado and Garcia de la Torre, provides the molecular dimensions of the aggregate structures. At about 0.10 mol/L the average stack size of the tetramers is estimated to 9 units per stack and for the monomer stacks to about 4 units. In both cases the rotational and translational diffusion coefficients correspond to a cylinder-symmetric molecule of almost equal length and diameter. At higher concentration (1.0 mol/L) of 5’-GMP the axial ratio increases to about 2.35 (stacked tetramers) and 3.4 (stacked monomers), respectively.
Introduction Numerous studies of purine and pyrimidine bases, nucleosides, and nucleotides in aqueous solution have been performed. They show that despite the lack of a linking sugar-phosphate backbone the bases tend to form vertical stacks, similar to the structure in and microcalorimetry3 have proven DNA or RNA. that nucleotides and nucleosides associate in water beyond the dimer stage. While the thermodynamic experiments provide information about the extent of intermolecular interaction, spectroscopic methods allow one to characterize the kind of interaction and the structure of the aggregates. The ordered structures can be observed by IR$ Raman,’+’ and NMR spectroscopyI0-ls of different nuclei. In aqueous solution vertical stacking of the planar bases dominates over hydrogen bonding interaction. Guanosine 5-monophosphate (5’-GMP) is unique among all other mononucleotides to self-aggregate to highly stable, regular structures. The basic unit is a planar tetramer (Figure 1). The stability is based on the specific conformation of the guanine base. Due to a perpendicular orientation of two hydrogen bond donors (N,-H, N2-H) and two hydrogen bond acceptors ( c 6 4 , N7), four guanine molecules can associate uniquely by hydrogen bonding. The formation of two hydrogen bonds per base explains the high stability of the tetrameric structure. X-ray diffraction studies of 5‘-GMP and 3’-GMP fibersI6J7provide strong evidence shows, for the specific structures. A comparison with p01y-G~~ that the tetramer units form vertical stacks, i.e. aggregates in which the aromatic plans of the tetramers are arranged parallel to each other. The importance of the individual hydrogen bonding interactions is demonstrated by comparison with modified nucleo-
tides.19 Recent studies suggest, that these tetrameric structures are also formed by guanine-rich DNA fragments?O They might play a significant role in the ordering process of linear chromosomes and in protein-DNA recognition. In acidic solution 5’-GMP self-aggregates to highly ordered s t r u c t ~ r e swhich ~ ~ ~ ’form anisotropic gels. In neutral and basic s o l u t i ~ nthe ~ *extent ~ ~ of vertical stacking decreases, mainly due to an increase of electrostatic repulsion, because the second proton dissociates from the phosphate group (pK, = 6.lZs).At low concentration and high temperature, respectively, the aggregation behavior of 5’-GMP is very similar to the other nucleotides. But at concentrations >0.15 mol/L and below a certain temperature, 5’-GMP forms the specific tetramer structures. Due to the higher negative charge of the monomer under neutral and slightly basic conditions, the stacking is limited. IR and NMR studies indicate the cooperativity of the aggregation process. The number of nonequivalent ~ i g n a l s , lstoichiometric ~.~~ studies,26and 13CNMR relaxation measurementsMled to the conclusion that the stacking is limited to the formation of octamers, dodecamers, and hexadecamers. The extent of ordering is very much dependent on the nature of the counterion. The ability of the alkali-metal cations to stabilize vertical tetramer s t a ~ k sdecreases ~ ~ - ~ in ~ the order K+ >> Na+, Rb+ >> Cs+,Li+. The influence of the different cations cannot be explained by their charge density. The tetrameric orientation of the 5’-GMP rather provides a cavity (see Figure I), and it is more likely that a size-specific metal complexation mechanism, well-known from crown ethers,27plays a dominate role. Therefore, the dimensions of the cavity in respect to the van der Waals radii of the different cations probably determine the
0022-365419212096-6790!§03.00/0 0 1992 American Chemical Society
Self-Aggregation of 5'-GMP
The Journal of Physical Chemistry, Vol. 96, No. 16, 1992 6791
emperarure
composed of spherical subunits. They give polynomial approximations to their numerical calculations which provide an analytical form for 0 and D for cylinder-symmetricmolecules with 2 Ip I 30, where p = L/d is the length to diameter ratio. Experiments on very short oligonucleotides4 have proven the effectiveness of this theory for cylindrical molecules with an axial ratio down to about 2. The purpose of this paper is to characterize the self-aggregation of Na-5'-GMP by studying the dynamics of the different structures, formed in aqueous solution. Measurements were performed over a broad concentration and temperature range. It will be shown, that larger stacks of the specific tetramer units of 5'-GMP are formed than previously assumed. In addition, the effect of NaCl on the aggregation process is described.
Experimental Section Material. The sodium salt of 5'-GMP was purchased from Pharma-Waldhof GmbH. The purity is >98% and the compound concentration was used without further purification. Solutions in doublediitilled water were filtered through a 0.22-ctm Millipore filter into fluorescence cells. The viscosity of the solution was determined in a micro-Ubbelohdeviscosimeter. The temperature was kept constant within fO.l OC by a thermostat. Depolarized IigM Scattering Experiments (DDIS). The DDLS apparatus is described in detail else~here.4~ The light source is an argon ion laser (Spectra-Physics, Model 165) in single-mode temperature with an output power between 200 and 450 mW. The sample concertrotion cell sits in a copper block, which is temperature controlled (Haake, Figure 1. (a) Schematic representation for the aggregation process of Model F3). The temperature is kept constant within 0.2 OC, 5'-GMP in solution. The triangular bars should indicate the influence measured by a Pt-resistance thermometer. The horizontal comof temperature and concentration on the average stack size and equilibponent of the scattered light is selected by a Glan-Thompson rium between monomer and tetramer units. The formation of tetramers polarizer with an extinction coefficient > 10'. The rotational is observed only for concentrations >0.15 mol/L 5'-GMP. (b) Oriencorrelation times of the different species vary between 100 ps and tation of the guanine bases in the planar, tetrameric structures formed 30 ns. The frequency broadening from the fast relaxation processes by 5'-GMP. ( l oblate ab
the magnitude of the scattering vector with n the refractive index of the solution, 8 the scattering angle, and A,, the wavelength of the incident light. In the DDLS experiment the time autocorrelation function of the horizontal component of the scattered light is observed. If the aggregates are rigid in the sense that intramolecular relaxation processes are slow compared to the overall motion, or if they do not change the laboratory-fixed polarizability significantly, then for a symmetric diffusor, Le., a cylinder-symmetric or ellipsoidal m0lecule5"~~
O2 is the mean-squared optical anisotropy of the aggregate
structure and 8, = 8, = e,, and e,,= e,, are the diagonal elements of the rotational diffusion tensor and D is the translational diffusion coefficient. f and g are the static and dynamic pair correlation factor?' respectively. For small molecules the reorientational motion is too fast to be observed by correlation techniques. Therefore, the DDLS experiment is performed in the frequency domain. The spectral density of the scattered light is given by the Fourier transform of eq 2. Assuming that the translational motion is much faster than the reorientation of the molecules, the contribution of q2D to the line width of the spectrum is negligible and
+mr r2 +
w2
