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Flat-coil probe for NMR spectroscopy of oriented membrane samples
JOURNALOF
MAGNETIC
RESONANCE
95,585-588
( 1991)
Flat-Coil Probe for NMR Spectroscopy of Oriented Membrane Samples B.BECHINGERAND Department
of Chemistry,
University
S.J. OPELLA
of Pennsylvania,
Philadelphia.
Pennsylvania
19104
Received May 14, 1991
A probe optimized for NMR studies of oriented lipid bilayer samples has been developed and demonstrated to be effective in single- and double-resonance experiments with a variety of nuclei. This probe is characterized by having a flat radiofrequency coil wrapped directly around a pair of square glass plates between which the actual sample is sandwiched. The very high efficiency of the probe results from the favorable filling factor, which in turn allows additional gains in efficiency through miniaturization of all capacitors, leads, and connectors. The square sample has a high ratio of area to circumference, reducing edge effects and improving the macroscopic orientation of the lipids. Neither lipid nor protein components of membranes are amenable to structural analysis by multidimensional solution NMR studies because of their slow overall reorientation rates. However, the structures of immobile peptides and proteins can be determined at atomic resolution by solid-state NMR methods when it is possible to orient the samples with respect to the magnetic field of the NMR spectrometer (I4). In favorable cases, spontaneous uniaxial orientation occurs due to interactions of the sample with the large external magnetic field. Magnetic alignment enabled the structure of the coat protein of the filamentous bacteriophage fd to be determined by solid-state NMR spectroscopy ( 1, 2). Some specific lipid mixtures and protein-containing membrane fragments have been found to orient in magnetic fields (5, 6). However, spontaneous sample alignment is exceptional and cannot be expected to work for most bilayer samples of interest. A commonly used and effective technique for aligning membranes involves the deposition of a mixture of lipids and proteins onto glass plates followed by application of pressure, shear, or centrifugal forces ( 7-1 I ) . A very high degree of orientation can be achieved when small amounts of lipids are placed between glass plates with relatively large overall surface area and the level of hydration subsequently reduced. Typically, a large number of rectangular glass plates are stacked in parallel so that a reasonable amount of material can be placed in the cylindrical coil of the probe (12-14). The preparation of large stacks of glass plates containing lipids has a number of serious limitations. It is laborious with difficulties arising from the lipid-containing material being extruded between the plates, the application of uniform pressure over the plates, and misalignment of the glass plates themselves. In addition, the overall filling factor of the coil is worse with a rectangular sample placed in a round coil. 585
0022-2364191 $3.00 Copyright 0 1991 by Academic F’rw, Inc. All rights of reproduction in any form reserved.
586
NOTES
The optimal sample configuration would be to place a large amount of material between two large round or (second best) square glass plates. The large surface area enhances the extent of orientation by providing a uniform environment for the lipids and the high ratio of circumference to area reduces edge effects. The use of a single pair makes it easier to apply uniform pressure and eliminates problems associated with the misalignment of stacks of glass plates. A high degree of orientation results when 20 mg of phospholipid is hydrated at 90% relative humidity and aligned between two 18 X 18 mm glass plates. A conventional solenoidal radiofrequency coil has weak spectroscopic performance with this sample, as can be seen in Fig. 1A. Inspired by Freeman’s (15) elegant theory of the filling factor, we prepared a “Flounder Corselette Coil” OF fish sandwich coil arrangement for a NMR probe, where a flat coil is wrapped directly around a pair of glass plates. The favorable filling factor results in a spectrum with much better signal-to-noise ratio as shown in the comparison of spectra in Figs. 1B and 1A. Technical data of both coils are presented in Table 1. In addition to the better receiver characteristics, the flat coil also exhibits better energy-transmitting properties as characterized by the 90” pulse length for the phosphorus nuclei and the power necessary to achieve decoupling of proton-phosphorous dipoiar interactions. Without extensive shimming the field homogeneity of both coils was sufficient for most purposes of solid-state NMR as is evident from the linewidth of the 3’P signal. The high efficiency of the probe allows the use of small fixed and variable capacitors which in combination with the miniaturization of leads and connectors results in even
I
100
I
0
-100
PPm FIG. 1. Proton-decoupled “P NMR spectra (60.89 MHz) of 15 pmol l-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) oriented between a pair of glass plates with an orientation of the bilayer normal parallel to the magnetic field. Spectrum A is retarded with a round coil, and spectrum B is recorded with a flat “Flounder Corselette CoiI” where the coil is directly wrapped around the sample. A pulse echo sequence with proton decoupling ( 150.4 1 MHz), quadrature detection, and phase cycling ( 18) was used (interpulse delay, 40 ps; recycling delay, 2s; number of scans, 80; memory size, 1024 points; spectral width, 50 kHz; line broadening, 100 Hz).
587
NOTES TABLE 1 Characteristics of the Flat and Round Coils Used in the “P-‘H Experiment of Oriented BiEavers
Double-Resonance
Coil shape
Round
Flat 19 X 19 mm*
Inner volume (mm3) Q value u/2 pulse length at 23 W ‘H decoupling power corresponding to 0.54 mT PCX’C linewidth (Hz) Peak height (arbitrary units) Signal/noise
d= 19mm I = 20 mm 5670 i50-200 20.5 ps 73 w 300-400 1.0 3
360 -120 6.5 ps 4w 300-350 4.1 13
better performance. An optimized double-resonance probe that combines the advantages of a flat coil with small capacitors and short wire connections is shown in Fig. 2. In some cases, for reasons of sensitivity, it may still be necessary to use multiple glass plates. We have found that a brick-shaped coil wrapped around these samples works well (16, 17). Other variations, such as fixing the coil to a goniometer and connecting it with flexible leads, should be feasible, enabling the orientation of both
FIG. 2. Flat-coil probe designed for “N- ‘H cross-polarization experiments with small fixed (American Technical Ceramics, New York) and variable (Johanson, Boonton, New Jersey) capacitors as we11as short leads in order to achieve enhanced sensitivity. The sample shown has the dimensions 18 X 18 mm.
588
NOTES
the sample and the coil to be varied while maintaining optimal filling factor. The improved spectroscopic performance which we obtained with two different coil shapes suggests that it is possible to adapt the coil to any pregiven sample as long as the radiofrequency circuit can be tuned at all frequencies of interest and the field homogeneity adjusted appropriately. ACKNOWLEDGMENTS We thank Russ Jacobs for valuable suggestions, Tai Lee for technical assistance in probe building, and Mike Mitchell for advice on the preparation of Fig. 2. This work was supported by grants ( ROl GM-2426614 and ROl GM-29754-10) from the National Institutes of Health. B.B. is supported by an EMBO long term fellowship ( ALTF 454-l 989). REFERENCES 1. T. A. CROSS AND S. J. OPELLA, .J. Mol. Biol. 182, 367 ( 1985). 2. S. J. OPELLA, P. L. STEWART, AND K. G. VALENTINE, Q. Rev. Biophys. 19,7 ( 1987). 3. S. J. OPELLA AND P. L. STEWART, in “Methods in Enzymology” (N. J. Oppenheimer and T. L. James, Eds.), Vol. 176, p. 242, Academic Press, San Diego, 1989. 4. L. E. CHIRLIAN AND S. J. OPELLA, New Polym. Matter 2, 279 ( 1990). 5. J. SEELIG, F. BORLE, ANLI T. A. CROSS, Biochim. Biophys. Acta 814, 195 (1985). 6. D. NEUGEBAUER, A. BLAUROCK, AND D. WORCESTER, FEBS Lett. 78,31 ( 1977). 7. P. C. JOST AND 0. H. GRI!=~ITH, Arch. B&hem. Biophys. 159,70 ( 1973). 8. J. K. BLASIE, M. ERECINSKA, S. SAMUELS, AND J. S. LEIGH, Biochim. Biophys. Acta 501, 33 ( 1978). 9. S. A. ASHER AND P. S. PERSHAN, Biophys. J. 27,393 (1979). IO. N. A. CLARK AND K. J. ROTHSCHILD, in “Methods in Enzymology” (L. Packer, Ed.), Vol. 88, p. 326, Academic Press, San Diego, 1982. 11. H. TANAKA AND J. H. FREED, J. Phys. Chem. 88,6633 (1984). 12. L. K. NICHOLSON, F. MOLL, T. E. MIXON, P. V. LOGRASSO, J. C. LAY, AND T. A. CROSS, Biochemistry 26,662l (1987). 13. B. A. CORNELL, F. SEPAROVIC, A. J. BALDASSI, AND R. SMITH, Biophys. J. 53,67( 1988). 14. A. W. HING, S. P. ADAMS, D. F. SILBERT, AND R. E. NORBERG, Biochemistry 29,4144 ( 1990). 15. R. FREEMAN, “A Handbook of NMR,” p. 220, Wiley, New York, 1988. 16. B. BECHINGER, Y. KIM, L. E. CHIRLIAN, J. GESELL, J.-M. NEUMANN, M. MONTAL, J. TOMtCH, M. ZASLOFF, AND S. J. OPELLA, J. Biomol. NMR 1, 167, ( 199 1). 17. K. SHON, P. SCHRADER, Y. KIM, B. BECHINGER, M. ZASLOFF, AND S. J. OPELLA, in “Biotechnology: Bridging Research and Applications” (D. Kamely, A. Chakrabarty, and S. Komguth, Eds.), p. 109, Kluwer Academic, Dordrecht, in press. 18. M. RANCE AND R. A. BYRD, J. Magn. Reson. 52,221 (1983).
MAGNETIC
RESONANCE
95,585-588
( 1991)
Flat-Coil Probe for NMR Spectroscopy of Oriented Membrane Samples B.BECHINGERAND Department
of Chemistry,
University
S.J. OPELLA
of Pennsylvania,
Philadelphia.
Pennsylvania
19104
Received May 14, 1991
A probe optimized for NMR studies of oriented lipid bilayer samples has been developed and demonstrated to be effective in single- and double-resonance experiments with a variety of nuclei. This probe is characterized by having a flat radiofrequency coil wrapped directly around a pair of square glass plates between which the actual sample is sandwiched. The very high efficiency of the probe results from the favorable filling factor, which in turn allows additional gains in efficiency through miniaturization of all capacitors, leads, and connectors. The square sample has a high ratio of area to circumference, reducing edge effects and improving the macroscopic orientation of the lipids. Neither lipid nor protein components of membranes are amenable to structural analysis by multidimensional solution NMR studies because of their slow overall reorientation rates. However, the structures of immobile peptides and proteins can be determined at atomic resolution by solid-state NMR methods when it is possible to orient the samples with respect to the magnetic field of the NMR spectrometer (I4). In favorable cases, spontaneous uniaxial orientation occurs due to interactions of the sample with the large external magnetic field. Magnetic alignment enabled the structure of the coat protein of the filamentous bacteriophage fd to be determined by solid-state NMR spectroscopy ( 1, 2). Some specific lipid mixtures and protein-containing membrane fragments have been found to orient in magnetic fields (5, 6). However, spontaneous sample alignment is exceptional and cannot be expected to work for most bilayer samples of interest. A commonly used and effective technique for aligning membranes involves the deposition of a mixture of lipids and proteins onto glass plates followed by application of pressure, shear, or centrifugal forces ( 7-1 I ) . A very high degree of orientation can be achieved when small amounts of lipids are placed between glass plates with relatively large overall surface area and the level of hydration subsequently reduced. Typically, a large number of rectangular glass plates are stacked in parallel so that a reasonable amount of material can be placed in the cylindrical coil of the probe (12-14). The preparation of large stacks of glass plates containing lipids has a number of serious limitations. It is laborious with difficulties arising from the lipid-containing material being extruded between the plates, the application of uniform pressure over the plates, and misalignment of the glass plates themselves. In addition, the overall filling factor of the coil is worse with a rectangular sample placed in a round coil. 585
0022-2364191 $3.00 Copyright 0 1991 by Academic F’rw, Inc. All rights of reproduction in any form reserved.
586
NOTES
The optimal sample configuration would be to place a large amount of material between two large round or (second best) square glass plates. The large surface area enhances the extent of orientation by providing a uniform environment for the lipids and the high ratio of circumference to area reduces edge effects. The use of a single pair makes it easier to apply uniform pressure and eliminates problems associated with the misalignment of stacks of glass plates. A high degree of orientation results when 20 mg of phospholipid is hydrated at 90% relative humidity and aligned between two 18 X 18 mm glass plates. A conventional solenoidal radiofrequency coil has weak spectroscopic performance with this sample, as can be seen in Fig. 1A. Inspired by Freeman’s (15) elegant theory of the filling factor, we prepared a “Flounder Corselette Coil” OF fish sandwich coil arrangement for a NMR probe, where a flat coil is wrapped directly around a pair of glass plates. The favorable filling factor results in a spectrum with much better signal-to-noise ratio as shown in the comparison of spectra in Figs. 1B and 1A. Technical data of both coils are presented in Table 1. In addition to the better receiver characteristics, the flat coil also exhibits better energy-transmitting properties as characterized by the 90” pulse length for the phosphorus nuclei and the power necessary to achieve decoupling of proton-phosphorous dipoiar interactions. Without extensive shimming the field homogeneity of both coils was sufficient for most purposes of solid-state NMR as is evident from the linewidth of the 3’P signal. The high efficiency of the probe allows the use of small fixed and variable capacitors which in combination with the miniaturization of leads and connectors results in even
I
100
I
0
-100
PPm FIG. 1. Proton-decoupled “P NMR spectra (60.89 MHz) of 15 pmol l-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) oriented between a pair of glass plates with an orientation of the bilayer normal parallel to the magnetic field. Spectrum A is retarded with a round coil, and spectrum B is recorded with a flat “Flounder Corselette CoiI” where the coil is directly wrapped around the sample. A pulse echo sequence with proton decoupling ( 150.4 1 MHz), quadrature detection, and phase cycling ( 18) was used (interpulse delay, 40 ps; recycling delay, 2s; number of scans, 80; memory size, 1024 points; spectral width, 50 kHz; line broadening, 100 Hz).
587
NOTES TABLE 1 Characteristics of the Flat and Round Coils Used in the “P-‘H Experiment of Oriented BiEavers
Double-Resonance
Coil shape
Round
Flat 19 X 19 mm*
Inner volume (mm3) Q value u/2 pulse length at 23 W ‘H decoupling power corresponding to 0.54 mT PCX’C linewidth (Hz) Peak height (arbitrary units) Signal/noise
d= 19mm I = 20 mm 5670 i50-200 20.5 ps 73 w 300-400 1.0 3
360 -120 6.5 ps 4w 300-350 4.1 13
better performance. An optimized double-resonance probe that combines the advantages of a flat coil with small capacitors and short wire connections is shown in Fig. 2. In some cases, for reasons of sensitivity, it may still be necessary to use multiple glass plates. We have found that a brick-shaped coil wrapped around these samples works well (16, 17). Other variations, such as fixing the coil to a goniometer and connecting it with flexible leads, should be feasible, enabling the orientation of both
FIG. 2. Flat-coil probe designed for “N- ‘H cross-polarization experiments with small fixed (American Technical Ceramics, New York) and variable (Johanson, Boonton, New Jersey) capacitors as we11as short leads in order to achieve enhanced sensitivity. The sample shown has the dimensions 18 X 18 mm.
588
NOTES
the sample and the coil to be varied while maintaining optimal filling factor. The improved spectroscopic performance which we obtained with two different coil shapes suggests that it is possible to adapt the coil to any pregiven sample as long as the radiofrequency circuit can be tuned at all frequencies of interest and the field homogeneity adjusted appropriately. ACKNOWLEDGMENTS We thank Russ Jacobs for valuable suggestions, Tai Lee for technical assistance in probe building, and Mike Mitchell for advice on the preparation of Fig. 2. This work was supported by grants ( ROl GM-2426614 and ROl GM-29754-10) from the National Institutes of Health. B.B. is supported by an EMBO long term fellowship ( ALTF 454-l 989). REFERENCES 1. T. A. CROSS AND S. J. OPELLA, .J. Mol. Biol. 182, 367 ( 1985). 2. S. J. OPELLA, P. L. STEWART, AND K. G. VALENTINE, Q. Rev. Biophys. 19,7 ( 1987). 3. S. J. OPELLA AND P. L. STEWART, in “Methods in Enzymology” (N. J. Oppenheimer and T. L. James, Eds.), Vol. 176, p. 242, Academic Press, San Diego, 1989. 4. L. E. CHIRLIAN AND S. J. OPELLA, New Polym. Matter 2, 279 ( 1990). 5. J. SEELIG, F. BORLE, ANLI T. A. CROSS, Biochim. Biophys. Acta 814, 195 (1985). 6. D. NEUGEBAUER, A. BLAUROCK, AND D. WORCESTER, FEBS Lett. 78,31 ( 1977). 7. P. C. JOST AND 0. H. GRI!=~ITH, Arch. B&hem. Biophys. 159,70 ( 1973). 8. J. K. BLASIE, M. ERECINSKA, S. SAMUELS, AND J. S. LEIGH, Biochim. Biophys. Acta 501, 33 ( 1978). 9. S. A. ASHER AND P. S. PERSHAN, Biophys. J. 27,393 (1979). IO. N. A. CLARK AND K. J. ROTHSCHILD, in “Methods in Enzymology” (L. Packer, Ed.), Vol. 88, p. 326, Academic Press, San Diego, 1982. 11. H. TANAKA AND J. H. FREED, J. Phys. Chem. 88,6633 (1984). 12. L. K. NICHOLSON, F. MOLL, T. E. MIXON, P. V. LOGRASSO, J. C. LAY, AND T. A. CROSS, Biochemistry 26,662l (1987). 13. B. A. CORNELL, F. SEPAROVIC, A. J. BALDASSI, AND R. SMITH, Biophys. J. 53,67( 1988). 14. A. W. HING, S. P. ADAMS, D. F. SILBERT, AND R. E. NORBERG, Biochemistry 29,4144 ( 1990). 15. R. FREEMAN, “A Handbook of NMR,” p. 220, Wiley, New York, 1988. 16. B. BECHINGER, Y. KIM, L. E. CHIRLIAN, J. GESELL, J.-M. NEUMANN, M. MONTAL, J. TOMtCH, M. ZASLOFF, AND S. J. OPELLA, J. Biomol. NMR 1, 167, ( 199 1). 17. K. SHON, P. SCHRADER, Y. KIM, B. BECHINGER, M. ZASLOFF, AND S. J. OPELLA, in “Biotechnology: Bridging Research and Applications” (D. Kamely, A. Chakrabarty, and S. Komguth, Eds.), p. 109, Kluwer Academic, Dordrecht, in press. 18. M. RANCE AND R. A. BYRD, J. Magn. Reson. 52,221 (1983).