19F-Labeling of Peptides Revealing Long-Range NMR Distances in

Letter pubs.acs.org/JPCL

19

F‑Labeling of Peptides Revealing Long-Range NMR Distances in Fluid Membranes

Stephan L. Grage,†,§ Xiaojun Xu,‡,§ Markus Schmitt,† Parvesh Wadhwani,† and Anne S. Ulrich*,†,‡ †

Institute of Biological Interfaces IBG-2, Karlsruhe Institute of Technology, P.O. Box 3640, 76021 Karlsruhe, Germany Institute of Organic Chemistry, Karlsruhe Institute of Technology, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany

‡

S Supporting Information *

ABSTRACT: NMR distance measurements lie at the heart of structural biology. However, long-range distances could not yet be detected in liquid−crystalline biomembranes, because dipolar couplings are partially averaged by the intrinsic molecular mobility. Using conformationally constrained 19F-labeled amino acids as reporter groups, we could more than double the accessible interatomic distance range by combining a highly sensitive solid-state multipulse 19F-NMR scheme with a favorable sample geometry. Two rigid 4F-phenylglycine labels were placed into the helical antimicrobial peptide PGLa embedded in fluid oriented membrane samples. A modified Carr−Purcell−Meiboom−Gill sequence yielded an intramolecular distance of 6.6 Å for the labels spanning one helix turn, and 11.0 Å was obtained when the labels spanned two turns. This approach should now also allow the characterization of conformational changes in membrane-active peptides and of oligomeric assemblies in a biologically relevant lipid environment. SECTION: Biomaterials, Surfactants, and Membranes

N

resolved spectra.14 Monofluoro reporter groups can yield fundamental structural parameters, such as intramolecular or intermolecular backbone−backbone distances, for which the labels have to be conformationally constrained (Figure 1). 4F-

MR is a versatile method for structural analysis, especially for biological applications in virtually all kinds of environments from liquid to soft to solid. Most applications are based on the ability to determine interatomic distances with high precision. However, NMR observables are sensitive to molecular mobility and can report only on time-averaged structures. Especially weak dipolar couplings, which are the basis for distance measurements, are already averaged by slow molecular motions. Hence, in solid-state NMR studies of “soft” materials, such as liquid crystals or biomembranes at ambient temperatures, dipolar couplings are significantly reduced, which impedes the measurement of long-range distances. For this reason, distances ≥ 10 Å have only been reported for genuinely solid materials (a detailed overview is in the Supporting Information). Any comparable measurements in fluid biomembranes have not been possible so far. Here, we demonstrate that the use of fluorine as a selective label with strong dipolar couplings is the key to enhance the distance range, so that peptides embedded in liquid−crystalline membranes can now be tackled. 19F-labeled amino acids offer an ideal compromise between the long-range FRET or ESR couplings (utilizing bulky fluorophores or electron spin labels) and the much shorter ranges accessible with nonperturbing NMR labels (such as 15N, 13C, or 2H). Several 19F-labeled amino acids have been previously designed and tailor-made to carry conformationally constrained CF3 groups for measuring local orientational parameters.1−8 The present study, however, is now aimed at 19F−19F distance measurements, for which monosubstituted side chains are required9−13 because the use of CF3 groups would lead to highly complex and poorly © 2014 American Chemical Society

Figure 1. Monofluoro substituted amino acids suitable for 19F-NMR distance measurements: (a) L-4F-Phg, (b) 4F-phenyl-3-cyclobutylglycine, and (c) L-2F-prop-2-enylglycine (n = 1) and L-3F-but-3enylglycine (n = 2).

Phenylglycine (4F-Phg) is used here, as shown in Figure 1a,15 and a related analogue (Figure 1b) has been recently prepared.9 In a similar way, direct H-bonding between equivalent side chains could be probed with 19F isosteres of glutamine or asparagine (Figure 1c).10,11 The geometry of peptide oligomers has been explored through distance constraints using 4Fphenylalanine and 5F-tryptophan,16 and interstrand proximities in peptide fibrils have been evaluated, for example, using a leucine analogue.17 However, as the side-chain torsion angles of these latter amino acid analogues are not known, accurate distance information about the peptide backbone or oligomeric structure is not available. To detect weak 19F−19F dipolar Received: October 16, 2014 Accepted: November 19, 2014 Published: November 19, 2014 4256

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interactions and interpret them correctly in terms of the underlying structure of the polypeptide backbone, we therefore explore here the use of the conformationally constrained 4FPhg as a first test case. To characterize membrane-active peptides and transmembrane proteins under quasi-native conditions, they need to be embedded in lipid bilayers, which are usually in a liquid− crystalline state. This condition is readily fulfilled, for example, in macroscopically oriented samples on solid supports, when the membranes are well hydrated, and when measurements are performed at temperatures above the gel-to-fluid lipid phase transition. However, so far, only orientational constraints11,13,18−28 and medium-range distances up to ∼5 Å17,29−33 could be acquired in such samples. Here, we were able to measure an 11 Å distance using 19F as a long-range probe on a conformationally constrained amino acid in combination with a favorable sample geometry and an optimized multipulse sequence. For demonstration, we used an amphiphilic α-helical peptide that is embedded in fluid lipid bilayers and exposed to substantial averaging due to molecular motion. The antimicrobial peptide PGLa was doubly 19Flabeled by substituting either Ala6 and Ala10 (referred to as PGLa6−10) or Ala6 and Ile13 (referred to as PGLa6−13) with 4FPhg. The 19F spin pairs were thus placed into a peptide with a well-known membrane-bound structure25 either four or seven residues apart, that is, spanning either one or two helix turns (see Figure 2a,b). For the interfluorine distance measurements, the labeled PGLa was reconstituted into dimyristoylphosphatidylcholine (DMPC) bilayers that were mechanically aligned on small glass plates and fully hydrated (see the Supporting Information for details of the sample preparation). NMR experiments were performed at 35 °C, where the DMPC bilayers are in the liquid−crystalline phase and the peptide undergoes rotational diffusion around the membrane normal. Under these conditions, PGLa is known to possess an additional mobility that can be described by an order parameter of Smol = 0.66, characterizing the distribution of motionally averaged helix orientations.25 The oriented sample was placed with the membrane normal parallel to the static spectrometer magnetic field (α = 0°), which has two advantages. In such an aligned sample, each labeled side chain gives rise to an intrinsically narrow signal, which helps to resolve the pure dipolar Carr−Purcell−Meiboom−Gill (CPMG) spectrum below. Furthermore, in the 0° orientation, the dipolar couplings are known to have a maximum value compared to any other orientation, being twice as strong as that for the main signals in a nonoriented “powder” sample (which correspond to α = 90° orientation; see the equation below). In the simple one-pulse 1H-decoupled 19F-NMR spectra of PGLa6−10 (Figure 2c) and PGLa6−13 (Figure 2d), however, the narrow doublet expected from the weak 19F−19F coupling is not visible. The presence of two lines rather reflects the different 19F chemical shifts of the two differently oriented labels. To address the dipolar interaction between the weakly coupled spins, we thus employed an optimized CPMG experiment.34,35 This multipulse train efficiently removes the contributions of the anisotropic 19F chemical shift as well as the heteronuclear 1H−19F dipolar coupling by repeated refocusing, while the homonuclear 19F−19F dipolar coupling still evolves. This way, even small doublets originating from the homonuclear 19F−19F dipolar couplings of distant 19F spin pairs can be revealed. In particular, the CPMG experiment is compatible with oriented samples as it does not rely on magic angle

Figure 2. Long-range 19F−19F distance measurement in oriented membranes in the fluid phase. The helical peptide PGLa was doubly labeled with 19F in two positions, either four or seven residues apart. The rigid reporter group 4F-Phg was incorporated in positions 6 and 10 in the analogue PGLa6−10 (a). In PGLa6−13, the two 19F labels were placed two helix turns apart in positions 6 and 13 (b). Standard onepulse 1H-decoupled solid-state 19F spectra of PGLa6−10 (c) and PGLa6−13 (d) were acquired in oriented DMPC bilayers at 35 °C. They exhibit two signals due to the different 19F chemical shifts of the differently oriented labels. Using the static Carr−Purcell−Meiboom− Gill (CPMG) experiment with xy8 phase cycling and 90−18090°−90° composite pulses, the purely dipolar spectrum reveals the 19F−19F coupling for PGLa6−10 (e) and PGLa6−13 (f). The CPMG spectra were fitted by a doublet line shape, with a splitting of 363 ± 17 (g) and 79 ± 12 Hz (h) for PGLa6−10 and PGLa6−13, respectively, corresponding to distances of 6.6 ± 0.3 and 11.0 ± 0.7 Å. Lorentzian line broadening of 330 and 115 Hz was used for fitting the spectra of PGLa6−10 and PGLa6−13, respectively.

spinning. This combination of experimental strategies allowed one to resolve the dipolar doublets in the CPMG spectra of PGLa6−10 (Figure 2e) and PGLa6−13 (Figure 2f). A splitting of 363 ± 17 Hz was obtained for the two 19F labels separated by four residues in PGLa6−10 (Figure 2g). Even the much weaker coupling of 79 ± 12 Hz of the labels that were two helix turns apart in PGLa6−13 was detectable (Figure 2h). In the case of a molecule undergoing rotational diffusion around the membrane normal, the dipolar splitting Δ can be converted into a distance r through the relationship 1 1 Δ = 3 b Smol (3 cos2 α − 1) (3 cos2 β − 1)r −3 2 2 where b = 122 kHz, α is the tilt angle of the membrane normal, β is the angle of the internuclear vector with respect to the membrane normal, and the order parameter Smol describes motional averaging due to additional wobble.36 To analyze our data, we can rely on the known orientation and mobility of PGLa as determined earlier.25 The tilt angle of the helix axis is 98° (relative to the membrane normal), and the alignment of 4257

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see numerous applications, especially for characterizing the molecular interface of membrane-bound homodimers, as they have a straightforward symmetry that helps to evaluate the data.

the helix is further described by an azimuthal angle of 115° (defined here between the membrane plane and the radial vector through the Cα of residue 12), and the order parameter is Smol = 0.66 ± 0.05. The interfluorine vectors are thus known to be tilted relative the membrane normal by approximately β = 95 ± 10° in the case of PGLa6−10 and by β = 95 ± 10° in the case of PGLa6−13, where the error estimates account for the limited knowledge of the exact helix geometry and spread of alignments found in previous studies.25 (The two β values are the same by coincidence.) With these alignments and a given sample orientation of α = 0°, the splitting in the case of PGLa6−10 of 363 ± 17 Hz corresponds to a 19F−19F distance of approximately 6.6 ± 0.3 Å, and the splitting of PGLa6−13 of 79 ± 12 Hz reflects a distance of 11.0 ± 0.7 Å. Assuming an ideal helix with a pitch of 5.4 Å, a diameter of the cylinder defined by the Cα atoms of 4.5 Å, an alignment of the Cα−Cβ bond with respect to the helix axis as used in Strandberg et al.,25 and a Cα−F distance of 5.64 Å,37 the respective theoretical distances are obtained as 7.8 and 10.8 Å for PGLa6−10 and PGLa6−13, respectively. Hence, it seems that the measured 19F−19F distance in the former sample does not agree so well with the predicted value as it was found to be shorter. We attribute this discrepancy to deviations from the assumed helix structure, possibly caused by interactions between the aromatic 4F-Phg side chains that are just one turn apart. On the other hand, the distance obtained experimentally for PGLa6−13 agrees very well with the predicted value, confirming that a long-range distance of more than 10 Å could be accurately measured even in the highly mobile environment of a fluid membrane. In summary, an internuclear distance of 11 Å was determined between two 19F labels spanning seven residues of a helix. This, to our knowledge unprecedented, NMR distance measurement in fluid membranes was possible by exploring four strategies: (i) Due to the high gyromagnetic ratio, 19F−19F dipolar couplings can bridge distances close to the maximum range that is accessible by NMR. (ii) Monofluorine substituted amino acid analogues as 19F labels lead to simple dipolar spectra with good resolution. (iii) Oriented samples aligned with their normal along the magnetic field direction yield narrow lines and select the optimum orientation (α = 0°) that gives the largest dipolar coupling in a rotationally averaged fluid membrane. (iv) The static CPMG experiment is very efficient as straightforward Fourier transform yields the spectrum of the pure dipolar coupling. (Under ideal circumstances without any reduction by scaling, and in practice, a moderate scaling factor has to be taken into account.14) Unlike the present approach, most alternative techniques for distance measurements by solid-state NMR rely on magic angle spinning of nonoriented samples. In that case, the membrane normal adopts all possible orientations α; hence, the reporter groups are not aligned in the same favorable way and cannot give rise to a uniform and maximum dipolar coupling. At this point, it should be noted that the advantage of oriented samples over nonoriented ones does not apply to immobilized molecules, for example, in frozen membranes that are lacking rotational diffusion (see the Supporting Information). However, rotational diffusion seems to be the rule even for oligomeric membrane-embedded peptides and large transmembrane proteins;18,19 therefore, biologically relevant conditions can be ideally met using the present strategy. It provides a new approach to substantially extend the accessible distance range in those cases, where such measurements are highly relevant but also most difficult to achieve due to mobility. We

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ASSOCIATED CONTENT

S Supporting Information *

Experimental methods, a detailed literature overview of longrange distance measurements using solid-state NMR and discussion of oriented versus nonoriented samples. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Address: Institute of Biological Interfaces IBG-2, Karlsruhe Institute of Technology, P.O. Box 3640, 76021 Karlsruhe, Germany. Author Contributions §

S.L.G. and X.X. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Andrea Eisele and Kerstin Scheubeck for help in the synthesis and Sergii Afonin for MALDI characterization of the peptides and discussion, a grant by the China Scholarship Council to Xiaojun Xu, and the Helmholtz Association (Biointerfaces program) for financial support.

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REFERENCES

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