Determination of the Relative Permittivity of Acetylcholinesterase

Dec 15, 2009 - aromatic residues which form the gorge. An exact value for the relative permittivity and/or local dielectric constant can contribute to...
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Determination of the Relative Permittivity of Acetylcholinesterase Angela S. F. Ramos and Simone Techert* Max-Planck-Institut f€ ur biophysikalische Chemie, IFG Structural Dynamics of (Bio)chemical Systems, 37077 G€ ottingen, Germany

ABSTRACT For a complete understanding of biomolecular functions, electrostatic interactions have to be characterized. These interactions are highly dependent on the relative permittivity of the surrounding medium, including the interior of proteins and binding site regions. However, determination of the relative permittivity is not trivial. Here, we present an experimental method for measuring such physical properties by determining the microenvironmental dielectric constants of the protein acetylcholinesterase, which are 5.79 ( 0.10 for the whole protein interior and 3.72 ( 0.33 for the active site/gorge region. Exact experimental dielectric constant values can improve predictions from molecular dynamics and ligand electrostatic steering simulations and consequently contribute to a better understanding of the fast hydrolysis of acetylcholinesterase. SECTION Kinetics, Spectroscopy

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nteractions between molecules are essential in all biological processes. Almost all of the biological interactions are of electrostatic nature and thus depend on the relative permittivity (RP) of the surrounding environment and the entities on which biochemical action takes place. The relative permittivity can be measured by investigating the local dielectric constant (LD) of the surrounding medium. Knowledge of the RP and/or LD allows a detailed understanding of the function of biomolecules.1 However, processes such as enzymatic reactions do not occur in solutions only but also in special microenvironments, that is, the active sites. As a consequence, one aim of our experimental efforts is the determination of the local dielectric constant and/or the RP of these microenvironments for a detailed understanding of the enzymatic functions. The determination of the RP, however, is not trivial. In the present work, we followed the route as discussed and monitored the changes of the RP for various solvents and acetylcholinesterase (AChE) by investigating the fluorescence properties of appropriate dyes as a function of solvent properties. We found values in agreement with the calculations mentioned in the literature.2 The active site/gorge RP is moderately lower than the average RP in the whole protein interior. This was expected because of the high content of aromatic residues which form the gorge. An exact value for the relative permittivity and/or local dielectric constant can contribute to more accurate predictions from molecular dynamics and ligand electrostatic steering simulations and, consequently, to a better understanding of the fast hydrolysis of acetylcholinesterase. According to the mechanism of solvatochroism, optically excited states of a chromophore interact with the surrounding solvent molecules, depending on their polarity and before returning to the ground state. These interactions are often reflected

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in the emission spectra of the dyes investigated. When the dipole moment of a fluorescent molecule is larger in the excited state than that in the ground state, the extent of solvation of the excited state strongly depends on the polarity of the varying solvent and increases the fluorescence in the Stokes shift with increasing solvent polarity. That is the case for the fluorophore tryptophan and dyes like the intramolecular electron donor/ electron acceptor compound N,N-dimethyl(4-pyren-1-phenyl)amine, PyDMA.3-6 The stronger the solute/solvent interaction, the bigger the Gibbs free energy of the solvation of the excited state, and the larger the red shift of the emission band of the fluorophore and the corresponding Stokes shift. Mathematically, this proportionality is described by the socalled Lippert-Mataga plot,7 where the gradient of the emission band maximum ν~max with respect to the solvent factor F is proportional to the dipole factor μ2/F3 by   ð1Þ ν~max ¼ ν~0max - 2=ðhcÞÞðμ2=F3 F Here, ν~0max is the emission band maximum for zero solvent factor, μ is the dipole moment of the fluorescing species, F denotes the cavity radius for the chromophore, h is Planck's constant, and c is the speed of light in a medium. The solvent factor F is defined as F=((ε - 1)/(2ε þ 1)) - ((n2 - 1)/(2n2 þ 1)), where ε is the dielectric constant of the surrounding medium and n is its index of refraction. The Lippert-Mataga plot is generally used to estimate the dipole moment of a fluorescing species. In this work, we use the reverse way. We apply the known Lippert-Mataga plot of the dye PyDMA and the tryptophan fluorophore to elucidate Received Date: November 12, 2009 Accepted Date: December 4, 2009 Published on Web Date: December 15, 2009

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DOI: 10.1021/jz900261z |J. Phys. Chem. Lett. 2010, 1, 417–419

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Figure 2. Measured fluorescence of AChE (solid curve) fitted by the log-normal fitted function (dotted curve).

The average fluorescence of the tryptophans distributed in the interior of the AChE shows an emission band maximum at 30752 cm-1 (Figure 2). The latter was fitted by applying a lognormal function. Since the refractive index of the solution is 1.333, the relative permittivity found is 5.79 ( 0.10. This means that for all emitting tryptophans, an average relative permittivity of 5.79 is found. Since the tryptophans are distributed in the whole interior of AChE, this value reflects an average relative permittivity of the tryptophan environment in the protein. The difference between the RP values determined from the PyDMA fluorescence and those from the tryptophans fluorescence shows that the active site/gorge region, where the PyDMA binds, contains more nonpolar residues than the region around the tryptophans, which are distributed in the whole interior of AChE. Analysis of the accessibility of the residues in the ee/AChE structure (1C2O) yields a result which is consistent with this RP difference. It was found that within the buried residues (e20% accessibility), 16.5% of the residues are ionic, 23.5% are noncharged polar, and 60.0% of the residues are nonpolar, while the gorge region is mainly nonpolar.9 The accessibility was calculated using the program Swiss-PDB viewer.10 Mainly two approximations limit the presented method. First, in the model used, the fluorophore is treated as a point dipole in the center of a spherical cavity. Second, the method of dipole moment determination is based on the classical treatment of the solvent as a dielectric continuum. Thus, the determination of the dielectric constant from the Lippert-Mataga plot always gives a relative permittivity which is an average over a sum of unknown relative permittivities. However, the treatment of a microenvironment around a chromophore as a dielectric continuum model has been successfully applied to the studies on the microstructure of preferential solvation.11 Some authors criticize the use of the Lippert-Mataga for H-bonding chromophores, like tryptophan, because the fluorescence shift would no longer depend on the dielectric constant.6,12 Nevertheless, the experiment of van Duuren,13 where the 1,2-dimethylindole and 1-methyl-2-phenylindole fluorescence (unable to form H-bonding networks) was compared with other H-bonding indoles in various solvents, including water, shows no significant difference between their Stokes shifts, indicating that the fluorescence shifts of the indoles depend mainly on the dielectric constant of the surrounding medium and not on special H-bonding. The RP found are consistent with the values calculated for protein RP, which are in the range of 2-10, but much more accurate.2

Figure 1. Lippert-Mataga plot for PyDMA (A) and pure tryptophan (B) in several solvents (closed square). The solvent factor F for the solution with the AChE-PyDMA complex and for the intrinsic fluorescence of AChE was calculated from the linear fit (open circle). The solvents of (A) are described in ref 7.

the unknown relative permittivity of the active site of AChE and its intramolecular environment. Thus, we use PyDMA as an extrinsic sensor and Trp as an intrinsic sensor for the relative permittivity of AChE, with the known Lippert-Mataga plot as the calibration curve. The spectral red shifts of PyDMA and tryptophan fluorescence maxima in different solvents, as a function of the solvent factor F, are summarized in Figure 1A and B. The maximum of the fluorescence bands of PyDMA shifts from 24594 cm-1 in n-hexane to 18437 cm-1 in formamide. This results in a linear dependence of the emission band maximum on the solvent factor (Figure 1A), according to ν~max = 24110 cm-1 - 6360 cm-1/F. The goodness of fit is reflected in the fit errors of 24110 ((360 cm-1) and 6360 cm-1 ((630 cm-1). As discussed in our previous work, PyDMA in complex with AChE has an emission maximum at 22176 cm-1 (450 nm), indicating a dielectric constant of 3.72 ( 0.33 for the PyDMA binding region. Since PyDMA is a competitive inhibitor for AChE,5 we can conclude that the determined relative permittivity corresponds to the active site/gorge region. The low relative permittivity value is comparable to that of benzene (relative permittivity = 2.3). This is expected for the high content of aromatic residues in the AChE gorge.8 Note that the amount of water molecules in the PyDMA-gorge complex may be different from the amount of water molecules in complex with other ligands. This would change the value of the relative permittivity significantly. The fluorescence of tryptophan changes from 32619 in toluene to 28713 cm-1 in water. The linearly fitted curve is presented by the equation ν~max = 33425 cm-1 - 6018 cm-1/F. Please note, that the experimental error ((130 cm-1) is bigger than the error bar reflecting the goodness of the fit, which is 33425 ((16 cm-1) and 6018 cm-1 ((27 cm-1).

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DOI: 10.1021/jz900261z |J. Phys. Chem. Lett. 2010, 1, 417–419

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The fast hydrolysis rate of AChE still remains an unresolved topic of interest. The mechanisms of electrostatic steering of the substrate and of product release have not yet completely been understood. To clarify this, several computational studies have been done. Some examples for these studies are the calculations of the electrostatic potential of AChE, which is important for the evaluation of the electrostatic steering of the substrate,14,15 the simulation of the diffusion in the active gorge,16,17 and the structural fluctuation simulations of the enzyme, which are important for the induced fit, for the close-open states of the bottleneck of the AChE gorge,18 and for the release of the product through the so-called “backdoor”. For these simulations, the Poisson-Boltzmann approach has mainly been used; however, its reliability depends heavily on the chosen value of the RP for the relevant protein region.2 Therefore, RP values are crucial for the accurate calculations of electrostatic potentials and consequently for the interactions and structural fluctuations. Nevertheless, as discussed by Schutz and Warshel,1 the calculation of RP is far from being trivial. Normally, the protein RP is assumed to be 2 or 4. The measured RP value of 5.79 for the interior of AChE can be applied to the computational studies, resulting in smaller values for the electrostatic interaction energy. For the simulation of the diffusion and electrostatic steering in the gorge, the RP value of 3.72 can be more appropriate.

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METHODS Achetylcholinesterase from Electrophorus electricus (ee/AChE) was purchased from Sigma (type V-S), purified as previously described,19 concentrated, and stored in 4 mM potassium phosphate buffer, pH 8.0, at T = -20 °C. The solvents used were purchased from Merck (spectroscopic grade). The solvent-sensitive dye N,N-dimethyl(4-pyren-1-phenyl)amine (PyDMA) was prepared as described earlier.4,5 L-tryptophan was purchased from Aldrich. Fluorescence spectra have been measured on a Fluorolog 3-22 (Jobin Yvon-Spex) coupled to a temperature controller (20 °C). Emission spectra were taken in 0.5 nm steps at an excitation wavelength of 337 nm for PyDMA and of 297 nm for tryptophan. The slits were adjusted to a 1 nm bandwidth for excitation and emission. The recorded spectra were automatically corrected in accordance with the wavelength dependence on lamp intensity, monochromator transmission, and photomultiplier response. Further details on the setup and the developed methodology can be found in refs 4-7.

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AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed.

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ACKNOWLEDGMENT A.R. thanks the DAAD (PKZ: A/01/16759).

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REFERENCES

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S.T. is grateful to DFG support (SFB 755).

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Schutz, C. N.; Warshel, A. What Are the Dielectric “Constants” of Proteins and How to Validate Electrostatic Models?. Proteins 2001, 44, 400–417.

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DOI: 10.1021/jz900261z |J. Phys. Chem. Lett. 2010, 1, 417–419