AFM visualization of mobile influenza A M2 molecules in planar bilayers.

We report the observation of influenza A M2 (M2) incorporated in a dipalmitoylphosphatidylcholine (DPPC) supported planar bilayer on mica, formed by use of a modified vesicle fusion method from proteoliposomes and visualized with contact mode atomic force microscopy. Incubation of proteoliposomes in a hyperosmotic solution and increased DPPC/M2 weight ratios improved supported planar bilayer formation by M2/DPPC proteoliposomes. M2's extra-bilayer domains were observed as particles estimated to protrude 1-1.5 nm above the bilayer surface and <4 nm in diameter. Particle density was 5-18% of the nominal tetramer density. Movement of observable M2 particles was independent of the probe tip. The mean lateral diffusion coefficient (D) of M2 was 4.4 +/- 1.0 x 10(-14) cm(2)/s. Eighty-two percent of observable particles were mobile on the observable timescale (D > 6 x 10(-15) cm(2)/s). Protein-protein interactions were also observed directly.

[1]  J. Zasadzinski,et al.  Formation of Tethered Supported Bilayers by Vesicle Fusion onto Lipopolymer Monolayers Promoted by Osmotic Stress. , 2000, Langmuir : the ACS journal of surfaces and colloids.

[2]  L. Tamm,et al.  Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers. , 1992, Biochimica et biophysica acta.

[3]  M. L. Wagner,et al.  Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker. , 2000, Biophysical journal.

[4]  R. Lal,et al.  Amyloid β protein forms ion channels: implications for Alzheimer's disease pathophysiology , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[5]  Carolyn R. Bertozzi,et al.  Control of cell adhesion and growth with micropatterned supported lipid membranes , 2001 .

[6]  A. Engel,et al.  The bacteriophage φ29 head–tail connector imaged at high resolution with the atomic force microscope in buffer solution , 1997, The EMBO journal.

[7]  M Gerstein,et al.  Volume changes on protein folding. , 1994, Structure.

[8]  B. Balcom,et al.  Lateral diffusion in model membranes is independent of the size of the hydrophobic region of molecules. , 1993, Biophysical journal.

[9]  P. Nollert,et al.  Lipid vesicle adsorption versus formation of planar bilayers on solid surfaces. , 1995, Biophysical journal.

[10]  A. Hay,et al.  Maturation of influenza A virus hemagglutinin--estimates of the pH encountered during transport and its regulation by the M2 protein. , 1992, Virology.

[11]  R. Lamb,et al.  Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus. , 2002, Biochemistry.

[12]  Fredrik Höök,et al.  Formation of Supported Lipid Bilayer Membranes on SiO2 from Proteoliposomes Containing Transmembrane Proteins , 2003 .

[13]  G Büldt,et al.  Force-induced conformational change of bacteriorhodopsin. , 1995, Journal of molecular biology.

[14]  Fredrik Höök,et al.  Intact Vesicle Adsorption and Supported Biomembrane Formation from Vesicles in Solution: Influence of Surface Chemistry, Vesicle Size, Temperature, and Osmotic Pressure† , 2003 .

[15]  J Wang,et al.  Structure of the transmembrane region of the M2 protein H+ channel , 2001, Protein science : a publication of the Protein Society.

[16]  B G de Grooth,et al.  High speed atomic force microscopy of biomolecules by image tracking. , 1999, Biophysical journal.

[17]  V. Zhdanov,et al.  Formation of supported membranes from vesicles. , 2000, Physical review letters.

[18]  Tiina Lehto,et al.  Observing structure, function and assembly of single proteins by AFM. , 2002, Progress in biophysics and molecular biology.

[19]  T. Ando,et al.  A high-speed atomic force microscope for studying biological macromolecules , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Ole G Mouritsen,et al.  Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy. , 2002, Biophysical journal.

[21]  J. Hoh,et al.  Direct Visualization of Vesicle−Bilayer Complexes by Atomic Force Microscopy , 2000 .

[22]  I. Reviakine,et al.  Formation of Supported Phospholipid Bilayers from Unilamellar Vesicles Investigated by Atomic Force Microscopy , 2000 .

[23]  T. Tjärnhage,et al.  From liposomes to supported, planar bilayer structures on hydrophilic and hydrophobic surfaces: an atomic force microscopy study. , 2000, Biophysical journal.

[24]  H. Mcconnell,et al.  Allogeneic stimulation of cytotoxic T cells by supported planar membranes. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Engel,et al.  Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. , 1999, Biophysical journal.

[26]  Z. Shao,et al.  Tris(hydroxymethyl)aminomethane (C4H11NO3) induced a ripple phase in supported unilamellar phospholipid bilayers. , 1994, Biochemistry.

[27]  A. Engel,et al.  Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy. , 1994, Biophysical journal.

[28]  J. Israelachvili.  Intermolecular and surface forces , 1985 .

[29]  C. Yip,et al.  Single molecule imaging of supported planar lipid bilayer--reconstituted human insulin receptors by in situ scanning probe microscopy. , 2002, Journal of structural biology.

[30]  G. Kochendoerfer,et al.  Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly. , 1999, Biochemistry.

[31]  R. Lamb,et al.  The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[32]  R. Lamb,et al.  Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers , 2003, Protein science : a publication of the Protein Society.

[33]  Z. Shao,et al.  The effect of deformation on the lateral resolution of atomic force microscopy , 1996, Journal of microscopy.

[34]  R. Lamb,et al.  Permeation and Activation of the M2 Ion Channel of Influenza A Virus* , 2000, The Journal of Biological Chemistry.

[35]  H. Butt,et al.  Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope. , 1991, Biophysical journal.

[36]  I. Gustafson,et al.  Planar lipid bilayers on solid supports from liposomes--factors of importance for kinetics and stability. , 1997, Biochimica et biophysica acta.

[37]  Shao,et al.  AFM tips: how sharp are they? , 1999, Journal of microscopy.

[38]  A. Engel,et al.  The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions. , 1997, Biophysical journal.