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* Max-Planck-Institut für Polymerforschung, 55128 Mainz, Germany; Universität Osnabrück, Fachbereich Physik, D-49069 Osnabrück, Germany; and Universität Osnabrück, Fachbereich Biologie/Chemie, D-49069 Osnabrück, Germany
Correspondence: Address reprint requests to Heinz-Jürgen Steinhoff, Fachbereich Physik Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany. Tel.: 49-541-9692675; E-mail: hsteinho{at}uos.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSACKNOWLEDGEMENTSREFERENCES |
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; Hubbell et al., 1998, 2000; Steinhoff, 2002). Here, natural amino acids at desired sites are replaced by cysteine residues which are then modified by nitroxide spin labels. This method has been successfully applied to define elements of the secondary and tertiary structure of membrane proteins, including solvent exposure (Hubbell et al., 1998), to determine the orientation and movements of individual segments of membrane proteins under physiological conditions or to characterize conformational changes that occur during protein function (Hubbell et al., 2000; Rink et al., 2000).
In the present report we apply and compare pulsed and continuous wave (cw) EPR methods to gather interresidue distance data and thereby local structural information of the membrane protein Na+/proline transporter PutP of Escherichia coli. The attachment of more than one spin label to a macromolecule leads to considerable changes of the cw EPR spectral shape if the interspin distance is <
2 nm. These changes are due to the dipolar interactions between the individual spin labels and provide a measure for the interspin distance. In the range from 0.4 nm to 1.1 nm the intensities of the half-field transitions (Eaton et al., 1993) measured at low temperature (T < 200 K) provide a reliable distance measure. Peak height ratios, second moments, deconvolution based methods, and fitting algorithms of cw EPR spectra were shown to allow distance determination within the range of 0.8–2.2 nm (Likhtenshtein, 1976; Rabenstein and Shin, 1995; Steinhoff et al., 1997; Radzwill et al., 2001; Altenbach et al., 2001). These methods have been successfully applied to determine structural models of integral membrane proteins and protein complexes and to follow their conformational changes (see, e.g., Perozo et al., 1998; Mchaourab and Perozo, 2000; Wegener et al., 2001). In the distance range above 1.6 nm the uncertainty increases because the separation of the dipolar broadening contribution from the natural line width becomes increasingly difficult. For interspin distances exceeding 2 nm cw EPR is not applicable anymore; here pulse EPR techniques are a powerful approach. These techniques include pulse electron-electron double resonance techniques (Milov et al., 1981) such as four-pulse double electron-electron resonance (DEER) (Pannier et al., 2000), the 2 + 1 pulse sequence (Kurshev et al., 1989), multiple-quantum EPR (Borbat and Freed, 1999), and single-frequency techniques for refocusing electron-electron couplings (Jeschke et al., 2000). A comparison between the different methods for characterizing the distances between pairs of spin labels applied to the structurally well characterized, water-soluble protein human carbonic anhydrase II can be found in Persson et al. (2001).
The aim of the present study is to apply DEER in combination with site-directed spin labeling for the determination of details of the so far unknown structure of the Na+/proline transporter PutP of E. coli. PutP is a member of the Na+/substrate symporter family (SSSF, TC 2.A.21) which currently comprises more than 200 similar proteins of pro- and eukaryotic origin (Reizer et al., 1994; Turk and Wright, 1997; Jung, 2001). These integral membrane proteins utilize the Na+ electrochemical gradient to drive the coupled uphill transport of a variety of substrates (sugars, amino acids, vitamins, osmolytes, ions, myo-inositol, urea, and water). For PutP of E. coli, gene fusion analyses, Cys accessibility studies, site-specific proteolysis, and site-directed spin labeling studies suggest a secondary structure model according to which PutP contains 13 transmembrane helices (TM) with the N-terminus located on the periplasmic side of the membrane and the C-terminus facing the cytoplasm (Jung et al., 1998a; Wegener et al., 2000) (compare also Fig. 1). As far as investigated, the 13-helix motif appears to be a special feature of the SSSF. Much attention has been paid to the identification of regions involved in the binding and translocation of Na+ and substrate (Jung, 2001; Pirch et al., 2002). Besides these studies, investigation of the structure and conformational dynamics of these transporters represents one of the current major challenges.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSACKNOWLEDGEMENTSREFERENCES |
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). For overexpression, the putP alleles were cloned into plasmid pTrc99a (Amann et al., 1988) using restriction endonucleases NcoI and HindIII. The resulting plasmids were transformed into E. coli WG170 (F- trp lacZ rpsL thi 
(putPA)101 proP219) (Stalmach et al., 1983). Cells were grown, membranes were prepared, and PutP was solubilized and purified by Ni-nitrilotriacetic acid affinity chromatography as described (Jung et al., 1998b). The eluted single Cys PutP molecules (in 50 mM KPi, pH 8.0, containing 300 mM KCl, 200 mM imidazole, 10% glycerol (v/v), and 0.04% ß-D-dodecylmaltoside (w/v)) were reacted with methanethiosulfonate spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (Toronto Research Chemicals, Toronto) at molar spin label/protein ratio of 10:1 at 4°C for 3 h (hereafter we will refer to the spin-labeled residue as R1). Afterwards, unbound label was removed by dialysis against 50 mM KPi, pH 8.0, containing 10% glycerol and 0.04% ß-D-dodecylmaltoside (w/v). The protein was reconstituted into proteoliposomes at a lipid/protein ratio of 20:1 (w/w) as described (Jung et al., 1998b). Finally, the proteoliposomes were washed twice with 50 mM KPi, pH 7.5, and resuspended in the same buffer to yield a PutP concentration of 30–80 µM. Proteoliposomes were frozen and stored in liquid N2 until use.
cw EPR measurements
cw EPR spectra were recorded with homemade X-Band EPR spectrometers equipped with a Bruker dielectric cavity for room-temperature (T 
293 K) measurements or with an AEG TE-103 (Bruker, Ettlingen, Germany) cavity for low-temperature (T 170 K) measurements. The magnetic field was adjusted using a Bruker B-NM 12 B-field meter; microwave power was determined using a HP 430C bolometer. The PutP samples were loaded into quartz capillaries with an inner diameter of 1 mm. At room temperature 10 scans with a scan width of 12.8 mT (microwave power <0.6 mW, modulation amplitude <0.33 mT) were averaged. At 170 K 10 or 20 scans (12.8 mT scan width, microwave power <0.4 mW, modulation amplitude 0.23 mT) were averaged. After 12-bit AD conversion the data were processed using a personal computer.
cw EPR spectra simulations
cw EPR powder spectra were simulated based on the method described previously (Steinhoff et al., 1997). The motion of proteins is strongly restricted for temperatures <200 K. Below that temperature the apparent hyperfine splitting was found to deviate no more than 2% from the rigid limit value determined at 80 K and the EPR line shape resembles that of a powder spectrum. To account for the natural line shape the calculated powder stick spectrum is convoluted by a superposition of a Gaussian and a Lorentzian. Dipolar interaction is considered by additional convolution with a Pake pattern (Steinhoff et al., 1997; Radzwill et al., 2001).
Four-pulse DEER measurements
Four-pulse DEER measurements were performed using a Bruker Elexsys 580 spectrometer equipped with a 3-mm split-ring resonator under conditions of strong overcoupling (Q 
100). The use of the 3-mm split-ring resonator increases sensitivity with respect to the 4-mm dielectric resonators used in our earlier work (Pannier et al., 2000) for the limited amount of protein samples and can provide a 2x larger microwave field amplitude at given incident power and quality factor. The measurements were performed at a temperature of 15 K to enhance sensitivity. With respect to measurements at 80 K, the number of accumulations in a given time decreases by a factor of 
due to the increase in the longitudinal relaxation time at 15 K. This is slightly overcompensated by the gain in Boltzmann population difference, which is a factor of 5.3, so that any increase in the phase memory time Tm directly translates to a sensitivity advantage. Before insertion into the probehead, the samples were shock-frozen in liquid nitrogen to avoid crystallization of water. The pulse sequence (
/2)
1-
1-()1-t'-()2-1 + 2 - t'-()1-2 -echo was used. (/2)- and ()-pulses had equal pulse lengths of 32 ns to assure equal excitation bandwidths. The interpulse delays were 1 = 200 or 400 ns and 2 = 1200 ns, except for the mutant A107R1/S223R1, where data for 1 = 240 ns, 2 = 2600 ns were also measured. The initial value of t' was 80 ns and the increment 8 ns. A phase cycle +x/-x was applied to the first pulse and the two signals were subtracted. The pump frequency 2 (typically 9.33 GHz) was set to the center of the resonator mode and the static magnetic field corresponded to pump excitation at the global maximum of the nitroxide spectrum. The observer frequency 1 (typically 9.395 GHz) was set to the local maximum at the low-field edge of the spectrum. Accumulation times for the data sets varied between 14 and 32 h.
Four-pulse DEER data analysis
Distances or distance distributions were obtained from the dipolar time evolution data by procedures described by Jeschke and co-workers (Jeschke, 2002; Jeschke et al., 2002). Because of the limited signal-to-noise ratio for these protein samples, we tried to keep the number of fit parameters as small as possible. Model distance distributions consisting of a homogeneous background concentration chom and a single Gaussian peak characterized by its mean distance rm, the standard deviation 
r, and an amplitude Ar were fitted to the data by minimization of the root mean square deviation using the shell factorization method (Jeschke et al., 2002) for signal simulation. Contributions of the Gaussian peak at distances r < 1.5 nm were dismissed, since spin pairs at such short distances cannot be excited by microwave pulses with pulse lengths tp 
16 ns (Jeschke, 2002). The data were also processed by a model-free approach based on a cross talk-corrected approximate Pake transformation, mapping of the dipolar frequency distribution to a distance distribution, and distance-domain smoothing (Jeschke et al., 2002) with a Gaussian filter of 0.4 nm width.
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RESULTS AND DISCUSSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSACKNOWLEDGEMENTSREFERENCES |
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). The results suggested that amino acid residues F45, S50, S54, W59, and M62 are part of TM II (cf. Fig. 1) whereas G39 and R40 on one side and A67 and G75 on the other side are located at membrane-water interfaces. TM III was suggested to be formed by residues S76 to G95. S71 and A107 were found to be located in water-exposed loops (loop3 and loop 4, respectively). Nitroxides side chains attached to positions 33 and 37 (loop 2) are on the cytoplasmic surface of the transporter but directed into an apolar environment. Binding of Na+ and/or proline to the transporter alters the mobility of the nitroxide group at positions 37 and 45. From these findings it was concluded that binding of ligands induces conformational alterations of PutP that involve at least part of TM II and the preceding cytoplasmic loop (loop L2). To further investigate the arrangement of cytoplasmic loops with respect to residue L37 the double Cys PutP derivatives L37C/A107C and L37C/D187C were prepared. EPR analyses of the spin-labeled samples were expected to provide information about distances between cytoplasmic loops L2 and L4 and between loops L2 and L6. In addition the doubly spin-labeled Cys PutP derivative A107C/S223C provides nitroxide positions on different sides of the lipid membrane (loops L4 and L7). According to the current model of PutP (Fig. 1) the interspin distance for this sample should exceed 4 nm.
cw EPR spectra (T = 293 K)
The room-temperature spectra of the single and double Cys PutP derivatives reacted with methane-thiosulfonate spin label are shown in Fig. 2. Differences in the S/N are due to different concentrations of the samples. The spectra of the single mutants L37R1 and A107R1 reveal intermediate motional restriction of the spin-label side chain as concluded from the apparent hyperfine splitting and the small line widths. This is in agreement with previous measurements and the proposed structure (Wegener et al., 2000) where these sites are located in the loops L2 and L4, respectively. In addition, accessibility measurements for water-soluble paramagnetic ions (Wegener et al., 2000) showed that the spin labels attached to positions 37 and 107 are close to the headgroup region of the bilayer or located in the water phase. The shape of the spectrum of the double mutant L37R1/A107R1 is in agreement with the superposition of the spectra of the two singly labeled samples (Fig. 2 b). The comparison of the spectra reveals that dipolar broadening is not observable in the cw EPR spectra of the double mutant at room temperature.
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, Fig. 1) position 187 is located in the very short loop L6 which accounts for a more restricted motion of this nitroxide. S223 is located in the periplasmic loop L7. Information on tertiary interactions of this residue has not been available so far. Cysteine accessibility studies and analysis of the effect of ligand binding of fluorescence-labeled PutP-S223C implicates the residue in proline-induced conformational alterations (T. Pirch and H. Jung, unpublished information).
cw EPR spectra (T < 200 K)
In none of the low-temperature cw EPR spectra of the spin-labeled double Cys PutP derivatives shown in Fig. 3 does the dipolar interaction between the unpaired electrons of the nitroxides lead to a clearly visible dipolar line broadening. EPR spectra simulations with magnetic tensors and line width parameters determined from singly labeled samples show only minor deviations from the experimental data (not shown). Best fittings were obtained with interspin distance values equal to or exceeding (2.0 ± 0.2) nm with the distance distribution width fixed to 0.3 nm. We have to conclude that the average interspin distance values for the three samples must be larger than 1.8 nm. Although exact distance values are not obtainable for these samples by means of the cw EPR method the results provide a lower limit for the interresidue distance values.
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1. For the control experiment on the singly labeled mutant L37R1 we expect an exponential decay of the signal governed by the homogeneous distribution of protein molecules in the sample. Indeed, the data (Fig. 4 a) can be fitted by an exponential decay corresponding to an approximate local protein concentration of 1.8 mM. In contrast, the signal for the doubly spin-labeled mutant L37R1/D187R1 deviates strongly from an exponential decay (Fig. 4 b). The deviation is due to a fast-decaying contribution close to t = 0 (arrow) that corresponds to preferred distances between the two labels shorter than 2.2 nm. To characterize the distance distribution in more detail and get an estimate for the reliability of the data, we also present results of two different data analysis procedures (Fig. 5, a and b). By using a model-free direct transformation of the data with the cross talk-corrected approximate Pake transformation (Jeschke et al., 2002) we obtain a distance distribution that is rather broad and may have a maximum at distances <1.8 nm that are not accessible by our experiments (solid line in Fig. 5 a). However, the cw EPR measurements show that the distribution function must significantly decrease for interspin distances <1.8 nm. A fit of a distance distribution consisting of a Gaussian peak (distance between the two labels in one protein molecule) and a homogeneous background (intermolecular distances) provides a distance distribution that is very close to the distribution obtained by direct transformation (solid line in Fig. 5 b). The extracted distance distribution did not significantly change when microwave pulses with lengths of 16 ns instead of 32 ns were used. Fits for several fixed mean distances rm and variation of r, Ar, and chom show that the data can still be described satisfactorily with mean distances up to 2.2 nm, but that the quality of the fit deteriorates strongly for rm > 2.2 nm (data not shown). The asymmetric width of the distribution was estimated to approach 0.8 nm, which suggests that the loop regions are very poorly ordered.
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The data for the doubly labeled mutant L37R1/A107R1 (Fig. 4 e) are also significantly nonexponential. Here the distance distribution extends to longer distances than for the mutant L37R1/D187R1 in the absence of NaCl (Fig. 5, c and d). We may safely conclude that the mean distance between the two labels is <2.5 nm. Addition of NaCl solution causes only a slight increase in the proton modulations for the mutant L37R1/A107R1 (data not shown) and no significant change in the distance distribution. The dipolar evolution is also essentially unchanged after subsequent addition of proline solution.
Yet a different behavior is observed for the doubly labeled mutant A107R1/S223R1 (Fig. 4 f). Again the data are significantly nonexponential, but in this case a slow oscillation corresponding to longer distances is observed, which is superimposed with strong proton modulations even in the absence of NaCl. To measure the larger distance with sufficient confidence we increased 2 to 2.6 µs (Fig. 6). The best fit (solid noiseless line) corresponds to a distance of 4.8 nm in agreement with the expectations for two residues on different sides of the membrane. The width of the Gaussian peak of 0.9 nm again indicates sizeable disorder in the loop regions, but note that this parameter is not very precisely defined at the given maximum dipolar evolution time t and signal-to-noise ratio. Fits with Gaussian peaks at several fixed mean distances and variation of r, Ar, and chom reveal that the absolute error of this distance is smaller than 0.8 nm.
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). The studies suggest that the hydrophilic loops between TMs IV and V and between X and XI are within 8 Å of each other. The current study provides first information on proximity relationships within PutP of E. coli and allows for the first time a determination of long-range distances within the transporter molecule. The DEER data are in good agreement with the recently proposed secondary structure model of PutP (Jung et al., 1998a; Wegener et al., 2000) (compare Fig. 7 and Fig. 1). The large mean distance (4.8 nm) between positions 107 and 223 strongly supports the idea of these positions being located on opposite sides of the membrane, whereas the much shorter distances determined between positions 37, 107, and 187 are in agreement with a location on the same side of the membrane. In addition, the large widths of distribution of distances between the different positions arise most likely from locations on flexible loop regions of the transporter. The shortest distance (1.8 nm) was determined between positions 37 and 187 located on the cytoplasmic loops between TMs I and II and TMs V and VI, respectively (Fig. 7). Although L37 itself is not important for PutP function, R40 in its vicinity plays a crucial role in coupling of Na+ and proline transport (Quick et al., 1999). Similarly, also D187 is important for an efficient coupling of the transport of both ligands (Quick and Jung, 1998). Both residues are highly conserved within the SSSF. Taken together with the DEER measurements, it can be speculated that R40 and D187 interact with each other during the transport cycle. Clearly, further analyses are required to substantiate this idea.
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; Jung, 2001; Pirch et al., 2002). In this study, the increase in proton modulations observed in the DEER measurements upon Na+ binding to PutP-L37R1/D187R1 suggests that one or both positions are exposed to a different environment most likely via a Na+-induced conformational change. An involvement of positions 37 and 187 in Na+-induced structural alterations is also suggested by the observed increase of the mean distance between these positions. In contrast to the 37/187 pair, Na+ and proline did not significantly influence the DEER signal of PutP-L37R1/A107R1. Ligand-induced distance changes in PutP-A107R1/S223R1 were not measured because the overall distance between positions 107 and 223 is too large and the DEER signal is approximately eightfold less sensitive to distance alterations than at shorter distances, as discussed, e.g., for the 37/187 pair. In conclusion, the studies demonstrate that pulse DEER techniques can be used to investigate the structure and structural alterations of integral membrane proteins. Four-pulse DEER has successfully been applied to obtain for the first time information on distances between amino acid positions in the tertiary structure of the Na+ proline transporter PutP. In addition, it is shown that Na+ binding leads to alterations of these distances.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSACKNOWLEDGEMENTSREFERENCES |
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This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 431-P10 (H.J.) and 431-P18 (H.J.S.)).
Submitted on September 15, 2003; accepted for publication December 5, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSACKNOWLEDGEMENTSREFERENCES |
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