17795
2005, 109, 17795-17798 Published on Web 09/02/2005
NMR Spectroscopic Study of Noble Gas Binding into the Engineered Cavity of HPr(I14A) from Staphylococcus carnosus Lydia Nisius, Max Stadler, Hans Robert Kalbitzer, and Eike Brunner* Institute of Biophysics and Physical Biochemistry, UniVersity of Regensburg, D-93040 Regensburg, Germany ReceiVed: July 18, 2005; In Final Form: August 16, 2005
Xenon binding into preexisting cavities in proteins is a well-known phenomenon. Here we investigate the interaction of helium, neon, and argon with hydrophobic cavities in proteins by NMR spectroscopy. 1H and 15 N chemical shifts of the I14A mutant of the histidine-containing phosphocarrier protein (HPr(I14A)) from Staphylococcus carnosus are analyzed by chemical shift mapping. Total noble gas induced chemical shifts, ∆, are calculated and compared with the corresponding values obtained using xenon as a probe atom. This comparison reveals that the same cavity is detected with both argon and xenon. Measurements using the smaller noble gases helium and neon as probe atoms do not result in comparable effects. The dependence of amide proton and nitrogen chemical shifts on the argon concentration is investigated in the range from 10 mM up to 158 mM. The average dissociation constant for argon binding into the engineered cavity is determined to be about 90 mM.
Introduction Specific interactions of noble gases with biomolecules were first observed for xenon by X-ray crystallography.1,2 Later crystallographic studies have shown the ability of other noble gases such as argon and krypton to bind into hydrophobic cavities of different size engineered into phage T4 lysozyme.3 Various liquid-state 129Xe NMR spectroscopic studies were carried out in order to investigate the interaction of xenon with proteins.4-7 Xenon is particularly well-suited for this purpose because it can be observed directly by NMR spectroscopy. Its chemical shift is highly sensitive to the local environment.8-10 Specific as well as nonspecific interactions of xenon with protein molecules in solution could be detected via 129Xe NMR spectroscopy.6 This method even allows to distinguish between different protein conformations.7 The sensitivity of 129Xe NMR spectroscopy can be enhanced dramatically by using so-called laser-polarized 129Xe.4,6,7 Furthermore, spin polarization can be transferred from laser-polarized 129Xe to nuclei located at the protein, thus enabling the observation of the so-called spin polarization induced nuclear Overhauser effect (SPINOE).11 This technique has recently been used to study hydrophobic pockets in lipid transfer proteins.12,13 Another possibility to study xenonprotein interactions is chemical shift mapping. In such experiments, xenon-induced chemical shift changes of nuclei like 1H, 13C, and 15N located at the protein are observed.4,13-15 The advantage of chemical shift mapping is the simplicity and robustness of this technique. In the present paper we investigate the interaction of helium, neon, and argon with a protein in solution. Compared to xenon, several properties make these atoms advantageous. Due to their smaller van der Waals radii (helium: 1.22 Å, neon: 1.54 Å, * Corresponding author. Phone: +49 941 943 2492, Fax: +49 941 943 2479, E-mail:
[email protected] 10.1021/jp0539371 CCC: $30.25
argon: 1.91 Å, xenon: 2.21 Å), they should be better suited to explore smaller cavities. Another important fact is that helium, neon, and argon do not form clathrate hydrates in aqueous solution in the relevant pressure-temperature range.16 Formation of clathrates is a limiting factor for the use of xenon as a probe atom. At room temperature, for example, the highest applicable pressure is about 1.5 MPa.17 Another benefit of helium and argon is their lower price compared to xenon. To study the binding of helium, neon, and argon into a hydrophobic cavity, the I14A mutant of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus carnosus was used as a model system. This mutant, HPr(I14A), exhibits an engineered cavity of ca. 3-4 Å diameter. The solution NMR structure (see Figure 1) as well as the complete spectral assignment of this mutant are known.15 The location of the engineered cavity and the ability of xenon to bind into this cavity has been investigated earlier.14,15 Experimental Section 15N labeled HPr(I14A) was prepared and purified as described by Go¨rler et al.18 The sample was kindly provided by Prof. Hengstenberg (Ruhr-Universita¨t Bochum, Germany). A highpressure sapphire tube (Saphikon, USA) equipped with a pressure-tight titanium valve similar to the one described by Baumer et al.19 was used in order to maintain a defined noble gas pressure within the sample tube. Pressures of the different gases were applied to adjust the noble gas concentration in the protein solution. Helium and neon pressures were directly applied to the sample tube. Defined amounts of argon were condensed into the tube from a calibrated volume using liquid nitrogen before the valve was closed. After the experiment, the leak-tightness of the sample tube was confirmed by a volumetric measurement of the amount of gas leaving the sample tube after opening the valve. The protein sample was degassed by at least
© 2005 American Chemical Society
17796 J. Phys. Chem. B, Vol. 109, No. 38, 2005
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Figure 1. Three-dimensional structure of HPr(I14A) and schematic representation of the cavity. Those amino acid residues exhibiting large argon-induced chemical shift changes are color coded. Dark gray: amino acid residues around the engineered cavity. Light gray: amino acid residues in the second strongly affected region. The computer program MOLMOL27 was used to design the figure.
three freeze and thaw cycles on a vacuum rack. The entire sample tube was evacuated after freezing the sample. Afterward, the sample was heated beyond 273 K. Dissolved gases then left the solution. This procedure was repeated three times. NMR spectra were obtained on a DMX-500 spectrometer (Bruker, Karlsruhe, Germany) at room temperature. Spectra were processed with the computer program XWINNMR (Bruker, Karlsruhe, Germany). 1H NMR resonances were referenced relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid, whereas 15N chemical shifts were referenced indirectly following the IUPAC recommendations.20 Peak picking and visualization were accomplished using the computer program AUREMOL.21 The concentration dependence of ∆ was fit to the function
c0 ∆n ) ∆max‚ c 0 + KD
Figure 2. Total noble gas induced chemical shifts for HPr(I14A) at room temperature as a function of the amino acid residue number for (a) helium (34 ( 2 mM), (b) neon (35 ( 2 mM), and (c) argon (32 ( 2 mM). The average values of ∆ (dashed) as well as the average values plus two standard deviations (dotted) are indicated by horizontal lines. The experimental error for ∆ amounts to 0.06 ppm.
(1)
using the software package Origin (Microcal, Northampton). Here, ∆n denotes the total argon-induced chemical shift of a specific amino acid at a defined argon concentration c0. ∆max is the saturation value of the argon-induced shift for this amino acid and KD is the dissociation constant. Results and Discussion To study the interaction of the noble gas atoms with HPr(I14A), 1H-15N heteronuclear single quantum coherence (HSQC) spectra were measured at various pressures up to about 10 MPa. Chemical shift data were analyzed using the formula introduced by Gro¨ger et al.14 in order to determine the total noble gas induced chemical shift (∆). Total helium-, neon-, and argoninduced chemical shifts are shown in Figure 2a, b, and c, respectively, for comparable concentrations of the probe atoms in solution. As can be seen, the effects of helium and neon are much smaller than those induced by argon. Figure 3 shows the argon-induced chemical shift changes for a selected region of the 1H-15N HSQC spectrum. Several residues exhibit significant argon-induced signal shifts in the 1H as well as the 15N dimension; other residues remain almost unshifted. Figure 4 shows the total argon-induced chemical shifts plotted as a function of the amino acid residue number compared with the xenon-induced chemical shifts at comparable concentrations in solution. As can be seen in Figure 4b, six residues are strongly influenced by argon (∆ ) 0.40-0.65 ppm). Strongly influenced means that the induced chemical shift is larger than the sum of the mean value and two standard deviations of ∆ calculated
Figure 3. Selected region from the 1H-15N HSQC spectrum (contour plot) of HPr(I14A). The spectra measured with argon (158 ( 8 mM, solid gray lines) and without argon (dashed black lines) at room temperature are superimposed.
for the entire molecule. These strongly influenced residues are indicated in Figure 1. They mainly cluster in two regions (amino acid residues 9-19 and 79-82). Furthermore, there is a third area (amino acid residues 51-59) showing lower but still significant induced chemical shifts. Comparison with our previously published results for xenon binding on HPr(I14A) (Figure 4a) reveals that residues affected by xenon are also influenced by argon. The argon-induced chemical shifts tend to be slightly smaller than those measured for the same concentration of xenon dissolved in the sample. Figure 4c shows the absolute values of the differences ∆Xe - ∆Ar in ppm for comparable probe atom concentrations. Obviously, amino acid residues in the areas 9-19 and 51-58 experience larger effects upon xenon binding compared to argon binding. In contrast,
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Figure 5. 1H-15N HSQC spectrum (contour plot) of the side chain amide groups of HPr(I14A). The spectra measured with argon (32 ( 2 mM, solid gray lines) and without argon (dashed black lines) at room temperature are superimposed.
Figure 4. Total noble gas induced chemical shifts for HPr(I14A) as a function of the amino acid residue number for (a) xenon (62 ( 3 mM; data are taken from ref 14) and (b) argon (57 ( 3 mM). The argon concentration was adapted to the xenon concentration used in ref 14. The average values of ∆ (dashed) as well as the average values plus two standard deviations (dotted) are indicated by horizontal lines. (c) Difference between the total argon- and xenon-induced chemical shifts as a function of the amino acid residue number. (d) Total argon-induced chemical shifts for the highest concentration applied in this study (158 ( 8 mM). The experimental error for ∆ amounts to 0.06 ppm.
the effect of both probe atoms upon the amino acid residues 79 and 82 is similar. At the maximum concentration applied in this study (158 ( 8 mM Ar, corresponding to 10.2 ( 0.5 MPa), argon-induced chemical shifts exceed those obtained for a xenon concentration of 62 ( 3 mM, corresponding to 1.4 MPa (see Figure 4d). Note that the latter pressure is close to the limiting pressure of about 1.5 MPa where clathrate formation starts at room temperature. Due to the lower solubility of argon compared to xenon (the Ostwald coefficients amount to 0.034322 and 0.099023 at room temperature, respectively), higher pressures are necessary in order to obtain equal concentrations in solution. However, purely pressure induced chemical shift changes can be neglected. At the highest pressure employed (about 10 MPa), such effects amount to less than 0.0028 ppm for 1H and 0.024 ppm for 15N,14,24 which corresponds to a maximum ∆ value of ca. 0.03 ppm. Argon-induced chemical shifts of size similar to the effects observed for the protein backbone were found for the side chain
amide groups. Again, helium and neon do not significantly influence the chemical shift of these signals. On the contrary, argon strongly influences their chemical shift (see Figure 5). As already stated for the backbone amide groups, purely pressure induced effects on the side chain amide groups can be excluded. The mean pressure coefficients for wild-type HPr amount to 0.075 and 0.0040 ppm for side chain 15N and 1H, respectively, at about 10 MPa,24 resulting in a ∆ value of ca. 0.08 ppm. As can be seen in Figure 5, the signals are mainly shifted in one direction and to a similar extent. The different behavior observed for the small gases helium and neon compared to argon and xenon might be attributed to two different properties: their polarizability and their size. The probe atoms argon and xenon exhibit higher polarizabilities. This obviously makes them capable of binding to the protein due to dipole-induced dipole and induced dipole-induced dipole interactions.3 Furthermore, their larger size may also be important for their ability to induce structural changes on the protein backbone which give rise to the observed chemical shift changes. The influence of size and polarizability on protein-ligand interactions was previously studied by X-ray crystallography.3 In that study, the polarizability of the noble gases could be correlated to their affinity to cavities and the occupancy of these cavities in mutants of T4 lysozyme. It was shown that argon, krypton, and xenon are able to bind into engineered cavities. In contrast, the electron density resulting from bound neon was too weak to refine structures.3 A similar tendency could be observed in recent molecular beam scattering experiments for water-noble gas interactions by Aquilanti et al.25 For the larger noble gases (argon, krypton, and xenon), the potential parameters for their interaction with water are larger than the values expected for pure van der Waals forces.26 Therefore, weak hydrogen bonds (”embryonic” hydrogen bonds) are postulated to be partially responsible for the interaction of these noble gases with water, in addition to van der Waals interactions. In contrast, pure van der Waals interactions occur for helium and neon. On the other hand, the size of the atoms may also be critical. The replacement of the sec-butyl moiety of isoleucine by the methyl group of alanine results in a tailor-made cavity for argon binding. With an approximate diameter of 3-4 Å,14 this cavity can perfectly host argon which has a diameter of 3.82 Å. Xenon (diameter 4.42 Å) is also able to bind into the engineered cavity which results in an expansion of the cavity.14,15 Helium and neon might be too small to induce any structural changes around
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Letters chemical shifts for these residues (Figure 4c). For comparison, it should be noted that dissociation constants observed for xenon binding into protein cavities are of the order of 9-20 mM.4,13 Conclusions In summary, it can be stated that argon binding into the engineered cavity of HPr(I14A) induces effects similar to those induced by xenon. This makes argon an interesting alternative to xenon in chemical shift mapping experiments. In contrast, helium and neon do not induce comparable effects due to their lower polarizability and/or smaller size. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (project Br 1278/12-1). L.N. thanks the Studienstiftung des deutschen Volkes (Bonn, Germany) for financial support. The authors thank Ms. Ingrid Cuno for carefully proofreading the manuscript. References and Notes
Figure 6. Total argon-induced chemical shifts for HPr(I14A) at room temperature as a function of the argon concentration shown for (a) isoleucine 9 (gray squares, KD ) 81 ( 10 mM), asparagine 10 (black squares, KD)89 ( 5 mM), alanine 14 (gray circles, KD ) 76 ( 9 mM), and alanine 19 (black circles, KD ) 106 ( 16 mM), and (b) asparagine 79 (gray triangles, KD ) 37 ( 4 mM) and serine 82 (black triangles, KD ) 53 ( 5 mM). Dissociation constants were determined by fitting the data to a two-site model. The experimental error for ∆ amounts to 0.07 ppm.
this cavity which are the reason for chemical shift changes of backbone 1H and 15N. Finally, the concentration dependence of the argon-induced chemical shift was studied (see Figure 6). The observed effects can all be described assuming a simple two-site exchange model, i.e., protein molecules rapidly exchange between the free and the argon-bound state. Dissociation constants can be determined by fitting the data to eq 1. For the amino acid residues 9, 10, 14, and 19, dissociation constants of 81 ( 10, 89 ( 5, 76 ( 9, and 106 ( 16 mM could be determined. These residues are close to the engineered cavity. That means argon binding into the cavity takes place with an average dissociation constant of about 90 mM. For the amino acid residues 79 and 82, which are not situated in direct proximity to the engineered cavity, dissociation constants of 37 ( 4 mM and 53 ( 5 mM have been determined. This may be due to the existence of a second binding site. The analysis of the protein structure with the computer program MOLMOL27 did not give evidence for this hypothesis. Another possible explanation would be the assumption that the protein exchanges between at least two different conformations even in the noble gas free sample. If the affinity of the noble gas atoms to these protein conformations is different, this selection will result in different apparent dissociation constants for the noble gas induced chemical shifts. Work is in progress to further analyze this phenomenon. Obviously, the residues 79 and 82 are more strongly influenced by argon compared to the other residues, which is in line with the observed small difference in argon- and xenon-induced
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