Skip to main content
SpringerLink
Log in

Uniaxial Diffusional Narrowing of NMR Lineshapes for Membrane Proteins Reconstituted in Magnetically Aligned Bicelles and Macrodiscs

  • Original Paper
  • Published:
Applied Magnetic Resonance Aims and scope Submit manuscript

Abstract

Structure and dynamics of membrane proteins can be effectively studied by oriented-sample solid-state nuclear magnetic resonance (NMR) techniques when the lipid bilayers are macroscopically aligned with respect to the main magnetic field. Magnetic alignment of the protein-containing membrane bilayer results from the negative susceptibility anisotropy of the lipid hydrocarbon interior yielding perpendicular sample alignment. At this orientation, while the uniformity of alignment represents an essential prerequisite for obtaining high-quality NMR spectra, further line narrowing is obtained by uniaxial motional averaging of the azimuthal parts of the chemical shift anisotropies and dipolar couplings. The motional averaging is brought about by uniaxial rotational diffusion of the protein molecules about the normal to the membrane surface, which is perpendicular to the magnetic field. Uniaxial averaging is efficient when the motion about the axis of alignment becomes sufficiently fast (on the timescale of the dipolar couplings and chemical shift anisotropies). Line narrowing under uniaxial rotation can be theoretically modeled using the stochastic Liouville equation. In this mini-review, we illustrate the method of uniaxial averaging for the relatively small Pf1 coat protein which exhibits excellent resolution in magnetically aligned bicelles due to its fast uniaxial diffusion and even superior resolution in large (30 nm) nanodiscs (macrodiscs) stabilized by a belt peptide. Spectra of Pf1 coat protein in polymer-stabilized macrodiscs, an alternative and more robust alignment media, are presented. We also report on preliminary spectra of a much larger protein—uniformly 15N labeled M1-M4 domain for the human acetylcholine receptor. While some spectral resolution is apparent, significantly broader linewidths emphasize the need for creating fast rotating discoidal membrane mimetics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano, Science 289(5480), 739 (2000)

    ADS  Google Scholar 

  2. V. Cherezov, D.M. Rosenbaum, M.A. Hanson, S.G.F. Rasmussen, F.S. Thian, T.S. Kobilka, H.-J. Choi, P. Kuhn, W.I. Weis, B.K. Kobilka, R.C. Stevens, Science 318(5854), 1258 (2007)

    ADS  Google Scholar 

  3. T. Warne, M.J. Serrano-Vega, J.G. Baker, R. Moukhametzianov, P.C. Edwards, R. Henderson, A.G.W. Leslie, C.G. Tate, G.F.X. Schertler, Nature 454(7203), 486 (2008)

    ADS  Google Scholar 

  4. S.H. White, W.C. Wimley, Annu. Rev. Biophys. Biomol. Struct. 28, 319 (1999)

    Google Scholar 

  5. A.G. Lee, Biochem. Soc. Trans. 39, 761 (2011)

    Google Scholar 

  6. A. Laganowsky, E. Reading, T.M. Allison, M.B. Ulmschneider, M.T. Degiacomi, A.J. Baldwin, C.V. Robinson, Nature 510(7503), 172 (2014)

    ADS  Google Scholar 

  7. P.J. Hamilton, A.N. Belovich, G. Khelashvili, C. Saunders, K. Erreger, J.A. Javitch, H.H. Sitte, H. Weinstein, H.J.G. Matthies, A. Galli, Nat. Chem. Biol. 10(7), 582 (2014)

    Google Scholar 

  8. C. Martens, R.A. Stein, M. Masureel, A. Roth, S. Mishra, R. Dawaliby, A. Konijnenberg, F. Sobott, C. Govaerts, H.S. McHaourab, Nat. Struct. Mol. Biol. 23(8), 744 (2016)

    Google Scholar 

  9. I.G. Denisov, S.G. Sligar, Nat. Struct. Mol. Biol. 23(6), 481 (2016)

    Google Scholar 

  10. F. Hagn, M. Etzkorn, T. Raschle, G. Wagner, J. Am. Chem. Soc. 135(5), 1919 (2013)

    Google Scholar 

  11. F. Hagn, G. Wagner, J. Biomol. NMR 61(3–4), 249 (2015)

    Google Scholar 

  12. K. Takeuchi, H. Arthanari, M.K. Imai, G. Wagner, I. Shimada, J. Biomol. NMR 64(2), 143 (2016)

    Google Scholar 

  13. N.J. Traaseth, L. Shi, R. Verardi, D.G. Mullen, G. Barany, G. Veglia, Proc. Natl. Acad. Sci. USA 106(25), 10165 (2009)

    ADS  Google Scholar 

  14. L.C. Shi, E.M.R. Lake, M.A.M. Ahmed, L.S. Brown, V. Ladizhansky, Biochimica et Biophysica Acta-Biomembranes 1788(12), 2563 (2009)

    Google Scholar 

  15. S.D. Cady, K. Schmidt-Rohr, J. Wang, C.S. Soto, W.F. DeGrado, M. Hong, Nature 463(7281), 689 (2010)

    ADS  Google Scholar 

  16. M. Sharma, M.G. Yi, H. Dong, H.J. Qin, E. Peterson, D.D. Busath, H.X. Zhou, T.A. Cross, Science 330(6003), 509 (2010)

    ADS  Google Scholar 

  17. S.H. Park, B.B. Das, F. Casagrande, Y. Tian, H.J. Nothnagel, M.N. Chu, H. Kiefer, K. Maier, A.A. De Angelis, F.M. Marassi, S.J. Opella, Nature 491(7426), 779 (2012)

    ADS  Google Scholar 

  18. M. Renault, M.P. Bos, J. Tommassen, M. Baldus, J. Am. Chem. Soc. 133(12), 4175 (2011)

    Google Scholar 

  19. S.L. Wang, R.A. Munro, L.C. Shi, I. Kawamura, T. Okitsu, A. Wada, S.Y. Kim, K.H. Jung, L.S. Brown, V. Ladizhansky, Nat. Methods 10(10), 1007 (2013)

    Google Scholar 

  20. C.H. Wu, A. Ramamoorthy, S.J. Opella, J. Magn. Reson. A 109, 270 (1994)

    ADS  Google Scholar 

  21. S.V. Dvinskikh, K. Yamamoto, A. Ramamoorthy, J. Chem. Phys. 125, 034507 (2006)

    ADS  Google Scholar 

  22. A.A. Nevzorov, S.J. Opella, J. Magn. Reson. 185, 59 (2007)

    ADS  Google Scholar 

  23. T. Gullion, J. Schaefer, J. Magn. Reson. 81, 196 (1989)

    ADS  Google Scholar 

  24. M.H. Levitt, in Encyclopedia of Nuclear Magnetic Resonance: vol. 9 (supplementary volume), ed. by D.M. Grant, R.K. Harris (Wiley, Chichester, 2002) pp. 165–195

  25. S.H. Park, B.B. Das, A.A. De Angelis, M. Scrima, S.J. Opella, J. Phys. Chem. B 114, 13995 (2010)

    Google Scholar 

  26. B.B. Das, H.J. Nothnagel, G.J. Lu, W.S. Son, Y. Tian, F.M. Marassi, S.J. Opella, J. Am. Chem. Soc. 134(4), 2047 (2012)

    Google Scholar 

  27. P.A. McDonnell, K. Shon, Y. Kim, S.J. Opella, J. Mol. Biol. 233(3), 447 (1993)

    Google Scholar 

  28. S.J. Opella, F.M. Marassi, J.J. Gesell, A.P. Valente, Y. Kim, M. Oblatt-Montal, M. Montal, Nat. Struct. Biol. 6(4), 374 (1999)

    Google Scholar 

  29. J. Wang, S. Kim, F. Kovacs, T.A. Cross, Prot. Sci. 10, 2241 (2001)

    Google Scholar 

  30. F.M. Marassi, S.J. Opella, Prot. Sci. 12(3), 403 (2003)

    Google Scholar 

  31. N.J. Traaseth, J.J. Buffy, J. Zamoon, G. Veglia, Biochemistry 45(46), 13827 (2006)

    Google Scholar 

  32. N.J. Traaseth, K.N. Ha, R. Verardi, L. Shi, J.J. Buffy, L.R. Masterson, G. Veglia, Biochemistry 47(1), 3 (2008)

    Google Scholar 

  33. R. Verardi, L. Shi, N.J. Traaseth, N. Walsh, G. Veglia, Proc. Natl. Acad. Sci. USA 108(22), 9101 (2011)

    ADS  Google Scholar 

  34. K. Yamamoto, M. Gildenberg, S. Ahuja, S.C. Im, P. Pearcy, L. Waskell, A. Ramamoorthy, Sci. Rep. 3, 2556 (2013)

    Google Scholar 

  35. N.J. Gleason, V.V. Vostrikov, D.V. Greathouse, R.E. Koeppe, Proc. Natl. Acad. Sci. USA 110(5), 1692 (2013)

    ADS  Google Scholar 

  36. A. Gayen, J.R. Banigan, N.J. Traaseth, Angew. Chem. Int. Edit. 52(39), 10321 (2013)

    Google Scholar 

  37. M.T. Ge, D.E. Budil, J.H. Freed, Biophys. J. 66(5), 1515 (1994)

    Google Scholar 

  38. M. Ge, J.H. Freed, Biophys. J. 74(2), 910 (1998)

    ADS  Google Scholar 

  39. B.G. Dzikovski, P.P. Borbat, J.H. Freed, Biophys. J. 87(5), 3504 (2004)

    ADS  Google Scholar 

  40. B.G. Dzikovski, P.P. Borbat, J.H. Freed, J. Phys. Chem. B 115(1), 176 (2011)

    Google Scholar 

  41. C.R. Sanders, J.H. Prestegard, Biophys. J. 58, 447 (1990)

    Google Scholar 

  42. C.R. Sanders, J.P. Schwonek, Biochem. 31, 8898 (1992)

    Google Scholar 

  43. R.R. Vold, R.S. Prosser, J. Magn. Reson. B 113, 267 (1996)

    Google Scholar 

  44. K.J. Glover, J.A. Whiles, G. Wu, N.-J. Yu, R. Deems, J.O. Struppe, R.E. Stark, E.A. Komives, R.R. Vold, Biophys. J. 81, 2163 (2001)

    Google Scholar 

  45. U.H.N. Durr, M. Gildenberg, A. Ramamoorthy, Chem. Rev. 112(11), 6054 (2012)

    Google Scholar 

  46. S.H. Park, S.J. Opella, J. Am. Chem. Soc. 132(36), 12552 (2010)

    Google Scholar 

  47. F. Scholz, E. Boroske, W. Helfrich, Biophys. J. 45(3), 589 (1984)

    Google Scholar 

  48. R.S. Prosser, J.S. Hwang, R.R. Vold, Biophys. J. 74, 2405 (1998)

    ADS  Google Scholar 

  49. R. Verardi, N.J. Traaseth, L. Shi, F. Porcelli, L. Monfregola, S. De Luca, P. Amodeo, G. Veglia, A. Scaloni, Biochimica et Biophysica Acta-Biomembranes 1808(1), 34 (2011)

    Google Scholar 

  50. S.H. Park, C. Loudet, F.M. Marassi, E.J. Dufourc, S.J. Opella, J. Magn. Reson. 193(1), 133 (2008)

    ADS  Google Scholar 

  51. M. Edidin, Annu. Rev. Biophys. Bioeng. 3, 179 (1974)

    Google Scholar 

  52. P.G. Saffman, M. Delbruck, Proc. Natl. Acad. Sci. USA 72, 3111 (1975)

    ADS  Google Scholar 

  53. R.J. Cherry, U. Muller, G. Schneider, FEBS Lett. 80(2), 465 (1977)

    Google Scholar 

  54. B.A. Lewis, G.S. Harbison, J. Herzfeld, R.G. Griffin, Biochemistry 24(17), 4671 (1985)

    Google Scholar 

  55. K.G. Valentine, D.M. Schneider, G.C. Leo, L.A. Colnago, S.J. Opella, Biophys. J. 49(1), 36 (1986)

    Google Scholar 

  56. S.H. Park, A.A. Mrse, A.A. Nevzorov, A.A. De Angelis, S.J. Opella, J. Magn. Reson. 178(1), 162 (2006)

    ADS  Google Scholar 

  57. M. Hong, T. Doherty, Chem. Phys. Lett. 432(1–3), 296 (2006)

    ADS  Google Scholar 

  58. S.D. Cady, C. Goodman, C.D. Tatko, W.F. DeGrado, M. Hong, J. Am. Chem. Soc. 129(17), 5719 (2007)

    Google Scholar 

  59. J.H. Spille, A. Zurn, C. Hoffmann, M.J. Lohse, G.S. Harms, Biophys. J. 100(4), 1139 (2011)

    ADS  Google Scholar 

  60. A.A. De Angelis, A.A. Nevzorov, S.H. Park, S.C. Howell, A.A. Mrse, S.J. Opella, J. Am. Chem. Soc. 126, 15340 (2004)

    Google Scholar 

  61. E.Y. Chekmenev, J. Hu, P.L. Gor’kov, W.W. Brey, T.A. Cross, A. Ruuge, A.I. Smirnov, J. Magn. Reson. 173(2), 322 (2005)

    ADS  Google Scholar 

  62. A. Marek, W.X. Tang, S. Milikisiyants, A.A. Nevzorov, A.I. Smirnov, Biophys. J. 108(1), 5 (2015)

    ADS  Google Scholar 

  63. S.H. Park, S. Berkamp, G.A. Cook, M.K. Chan, H. Viadiu, S.J. Opella, Biochemistry 50(42), 8983 (2011)

    Google Scholar 

  64. V.S.K. Ramadugu, G.M. Di Mauro, T. Ravula, A. Ramamoorthy, Chem. Commun. 53(78), 10824 (2017)

    Google Scholar 

  65. T. Ravula, S.K. Ramadugu, G. Di Mauro, A. Ramamoorthy, Angew. Chem. Int. Edit. 56(38), 11466 (2017)

    Google Scholar 

  66. J. Radoicic, S.H. Park, S.J. Opella, Biophys. J. 115, 22 (2018)

    ADS  Google Scholar 

  67. R. Kubo, J. Math. Phys. 4(2), 174 (1963)

    ADS  Google Scholar 

  68. R. Kubo, J. Phys. Soc. Jpn. Suppl. 26, 1 (1969)

    ADS  Google Scholar 

  69. J.H. Freed, in Electron Spin Relaxation in Liquids, ed. by L.T. Muus, P.W. Atkins (Plenum Press, New York, 1972) pp. 387–410

  70. D.J. Schneider, J.H. Freed, Adv. Chem. Phys. 73, 387 (1989)

    Google Scholar 

  71. A.A. Nevzorov, J.H. Freed, J. Chem. Phys. 112(3), 1413 (2000)

    ADS  Google Scholar 

  72. A.A. Nevzorov, J. Magn. Reson. 209, 161 (2011)

    ADS  Google Scholar 

  73. A.A. Nevzorov, J. Phys. Chem. B 115(51), 15406 (2011)

    Google Scholar 

  74. W.H. Steeb, Matrix Calculus and Kronecker Product with Applications and C++ Programs (World Scientific, Singapore, 1997)

    MATH  Google Scholar 

  75. A.A. Nevzorov, A.A. De Angelis, S.H. Park, S.J. Opella, in NMR Spectroscopy of Biological Solids, ed. by A. Ramamoorthy (Marcel Dekker, New York, 2005) pp. 177–190

  76. R. Kubo, J. Phys. Soc. Jpn. 17, 1100 (1962)

    ADS  Google Scholar 

  77. A. Abragam, The Principles of Nuclear Magnetism (Oxford University Press, London, 1961)

    Google Scholar 

  78. L.J. Edwards, D.V. Savostyanov, A.A. Nevzorov, M. Concistrè, G. Pileio, I. Kuprov, J. Magn. Reson. 235, 121 (2013)

    ADS  Google Scholar 

  79. A.A. Nevzorov, J. Magn. Reson. 249, 9 (2014)

    ADS  Google Scholar 

  80. E. Meirovitch, Z.C. Liang, J.H. Freed, Solid State Nucl. Magn. Reson. 89, 35 (2018)

    Google Scholar 

  81. G. Cornilescu, A. Bax, J. Am. Chem. Soc. 122, 10143 (2000)

    Google Scholar 

  82. A.A. Nevzorov, S. Moltke, M.P. Heyn, M.F. Brown, J. Am. Chem. Soc. 121, 7636 (1999)

    Google Scholar 

  83. A.A. De Angelis, S.C. Howell, A.A. Nevzorov, S.J. Opella, J. Am. Chem. Soc. 128(37), 12256 (2006)

    Google Scholar 

  84. A.A. Nevzorov, S.J. Opella, J. Magn. Reson. 160(1), 33 (2003)

    ADS  Google Scholar 

  85. Y.Y. Yin, A.A. Nevzorov, J. Magn. Reson. 212, 64 (2011)

    ADS  Google Scholar 

  86. A. Bielecki, A.C. Kolbert, H.J.M. de Groot, R.G. Griffin, M.H. Levitt, Adv. Magn. Reson. 14, 111 (1990)

    Google Scholar 

  87. V.V. Gurevich, E.V. Gurevich, Trends Pharmacol. Sci. 29(5), 234 (2008)

    Google Scholar 

  88. V.V. Gurevich, E.V. Gurevich, Trends Neurosci. 31(2), 74 (2008)

    Google Scholar 

  89. D.S. Thiriot, A.A. Nevzorov, L. Zagyanskiy, C.H. Wu, S.J. Opella, J. Mol. Biol. 341, 869 (2004)

    Google Scholar 

  90. S.H. Park, F.M. Marassi, D. Black, S.J. Opella, Biophys. J. 99(5), 1465 (2010)

    ADS  Google Scholar 

  91. V. Bondarenko, D. Mowrey, T. Tillman, T.X. Cui, L.T. Liu, Y. Xu, P. Tang, Biochimica et Biophysica Acta-Biomembranes 1818(5), 1261 (2012)

    Google Scholar 

  92. V. Bondarenko, D.D. Mowrey, T.S. Tillman, E. Seyoum, Y. Xu, P. Tang, Biochimica et Biophysica Acta-Biomembranes 1838(5), 1389 (2014)

    Google Scholar 

  93. N. Das, D.T. Murray, T.A. Cross, Nat. Protocols 81(11), 2256 (2013)

    Google Scholar 

  94. F. Delaglio, S. Grzesiek, G.W. Vuister, G. Zhu, J. Pfeifer, A. Bax, J. Biomol. NMR 6, 277 (1995)

    Google Scholar 

  95. W.X. Tang, A.A. Nevzorov, J. Magn. Reson. 212(1), 245 (2011)

    ADS  Google Scholar 

  96. L.C. Welsh, M.F. Symmons, D.A. Marvin, Acta Crystallogr. D Biol. Crystallogr. 56, 137 (2000)

    Google Scholar 

  97. D.S. Thiriot, A.A. Nevzorov, S.J. Opella, Protein Sci. 14, 1064 (2005)

    Google Scholar 

  98. A. Goldbourt, B.J. Gross, L.A. Day, A.E. McDermott, J. Am. Chem. Soc. 129(8), 2338 (2007)

    Google Scholar 

  99. W.X. Tang, R.W. Knox, A.A. Nevzorov, J. Biomol. NMR 54(3), 307 (2012)

    Google Scholar 

  100. E.O. Awosanya, A.A. Nevzorov, Biophys. J. 114, 1 (2018)

    Google Scholar 

  101. A.A. Nevzorov, J. Am. Chem. Soc. 130, 11282 (2008)

    Google Scholar 

  102. J. Katsaras, T.A. Harroun, J. Pencer, M.P. Nieh, Naturwissenschaften 92(8), 355 (2005)

    ADS  Google Scholar 

  103. R. Soong, P.M. Macdonald, Langmuir 25(1), 380 (2009)

    Google Scholar 

  104. R. Bruschweiler, F.L. Zhang, J. Chem. Phys. 120(11), 5253 (2004)

    ADS  Google Scholar 

  105. T. Short, L. Alzapiedi, R. Brueschweiler, D. Snyder, J. Magn. Reson. 209(1), 75 (2011)

    ADS  Google Scholar 

  106. M. Orwick-Rydmark, J.E. Lovett, A. Graziadei, L. Lindholm, M.R. Hicks, A. Watts, Nano Lett. 12(9), 4687 (2012)

    ADS  Google Scholar 

  107. J.M. Dorr, S. Scheidelaar, M.C. Koorengevel, J.J. Dominguez, M. Schafer, C.A. van Walree, J.A. Killian, Eur. Biophys. J. Biophys. Lett.rs 45(1), 3 (2016)

    Google Scholar 

  108. R.F. Zhang, I.D. Sahu, L.S. Liu, A. Osatuke, R.G. Corner, C. Dabney-Smith, G.A. Lorigan, Biochimica et Biophysica Acta-Biomembranes 1848(1), 329 (2015)

    Google Scholar 

  109. J.O. Colon-Saez, J.L. Yakel, J. Physiol. Lond. 589(13), 3163 (2011)

    Google Scholar 

  110. N. Unwin, Nature 373, 37 (1995)

    ADS  Google Scholar 

  111. N. Unwin, J. Mol. Biol. 346, 967 (2005)

    Google Scholar 

  112. M. Montal, S.J. Opella, Biochimica et Biophysica Acta-Biomembranes 1565(2), 287 (2002)

    Google Scholar 

  113. N. Kouvatsos, P. Giastas, D. Chroni-Tzartou, C. Poulopoulou, S.J. Tzartos, Proc. Natl. Acad. Sci. USA 113(34), 9635 (2016)

    Google Scholar 

  114. J.P. Changeux, Nat. Rev. Neurosci. 11(6), 389 (2010)

    Google Scholar 

  115. R. Peters, R. Cherry, Proc. Natl. Acad. Sci. USA 79, 4317 (1982)

    ADS  Google Scholar 

  116. R.A. Shapiro, A.J. Brindley, R.W. Martin, J. Am. Chem. Soc. 132(33), 11406 (2010)

    Google Scholar 

  117. S. Isabettini, S. Massabni, J. Kohlbrecher, L.D. Schuler, P. Walde, M. Sturm, E.J. Windhab, P. Fischer, S. Kustert, Langmuir 33(34), 8533 (2017)

    Google Scholar 

  118. S. Isabettini, M. Liebi, J. Kohlbrecher, T. Ishikawa, P. Fischer, E.J. Windhab, P. Walde, S. Kuster, Phys. Chem. Chem. Phys. 19(17), 10820 (2017)

    Google Scholar 

Download references

Acknowledgements

A.A.N. is indebted to Professor Jack H. Freed for the invaluable postdoctoral training in his laboratory, without which this work would have been impossible. Supported by the National Science Foundation grant CHE-1508400.

Author information

Authors and Affiliations

  1. Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA

    Deanna M. Tesch, Zhaleh Pourmoazzen, Emmanuel O. Awosanya & Alexander A. Nevzorov

  2. Department of Chemistry, Shaw University, 118 E South St, Raleigh, NC, 27601, USA

    Deanna M. Tesch

Authors
  1. Deanna M. Tesch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhaleh Pourmoazzen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Emmanuel O. Awosanya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander A. Nevzorov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander A. Nevzorov.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tesch, D.M., Pourmoazzen, Z., Awosanya, E.O. et al. Uniaxial Diffusional Narrowing of NMR Lineshapes for Membrane Proteins Reconstituted in Magnetically Aligned Bicelles and Macrodiscs. Appl Magn Reson 49, 1335–1353 (2018). https://doi.org/10.1007/s00723-018-1056-4

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00723-018-1056-4

Search

Navigation