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| The EMBO Journal
(1999) 18, 4981–4987, doi:10.1093/emboj/18.18.4981 |
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| High resolution AFM topographs of the Escherichia coli water channel aquaporin Z |
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| Simon Scheuring1, Philippe Ringler1, Mario Borgnia2, Henning Stahlberg1, Daniel J. Müller1, Peter Agre2 and Andreas Engel1 |
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1 M.E.Müller Institute for Structural Biology at the Biozentrum, University of Basel, Basel CH-4056, Switzerland 2 Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA To whom correspondence should be addressed Andreas Engel, aengel@ubaclu.unibas.ch Received 1 June 1999; Revised 19 July 1999; Accepted 20 July 1999. |
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| Abstract |
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Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning  -helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p4212 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change. |
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| Keywords: atomic force microscopy, bacterial water channel, loops, sidedness, volume |
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