The Atomic Force Microscope (AFM) is a 20 years old instrument that has proven valuable for the ultrastructural characterization of a number of systems. Its ability to record sub-nanometre resolution micrographs while operating in physiologic environments has unveiled the structure of many biological molecules and has also shown their dynamic behaviour. This and related techniques have proven very efficient in studying the topography of biological and reconstructed membranes, to the point of showing at high resolution the location of membrane proteins and their motion in real-time.[1] A younger technique derived from AFM is Single Molecule Force Spectroscopy. Here, mechanical force is applied to a single molecule thanks to a microfabricated probe to measure its stiffness or to apply tension to it. When applied to proteins, soluble or in a membrane, this technique can achieve and characterize the directional unfolding of single protein molecules, in the environment of choice.[2] The more common AFM imaging capabilities can be coupled with force spectroscopy measurements in order to, for example, first image a membrane and then pull out a protein from it, characterizing the unfolding and the pulling process.[3] In this contribution, we will present the main features of the AFM and force-spectroscopy technique and the results of its use for protein unfolding obtained at our research group. [1] Shevchuk, A.I., G.I. Frolenkov, D. Sánchez, P.S. James, N. Freedman, M.J. Lab, R. Jones, D. Klenerman, and Y.E. Korchev, Imaging Proteins in Membranes of Living Cells by High-Resolution Scanning Ion Conductance Microscopy. Angew Chem Int Ed, 2006. Published Online: 28 Feb 2006. [2] Samorì, B., G. Zuccheri, and P. Baschieri, Protein unfolding and refolding under force: methodologies for nanomechanics. Chemphyschem, 2005. 6(1): p. 29-34. [3] Oesterhelt, F., D. Oesterhelt, M. Pfeiffer, A. Engel, H.E. Gaub, and D.J. Muller, Unfolding pathways of individual bacteriorhodopsins. Science, 2000. 288(5463): p. 143-6.

"Atomic Force Microscopy and Single-Molecule AFM Force Spectroscopy for the Characterization of Membrane Proteins".

ZUCCHERI, GIAMPAOLO;VALLE, FRANCESCO;SANDAL, MASSIMO;SAMORI', BRUNO
2006

Abstract

The Atomic Force Microscope (AFM) is a 20 years old instrument that has proven valuable for the ultrastructural characterization of a number of systems. Its ability to record sub-nanometre resolution micrographs while operating in physiologic environments has unveiled the structure of many biological molecules and has also shown their dynamic behaviour. This and related techniques have proven very efficient in studying the topography of biological and reconstructed membranes, to the point of showing at high resolution the location of membrane proteins and their motion in real-time.[1] A younger technique derived from AFM is Single Molecule Force Spectroscopy. Here, mechanical force is applied to a single molecule thanks to a microfabricated probe to measure its stiffness or to apply tension to it. When applied to proteins, soluble or in a membrane, this technique can achieve and characterize the directional unfolding of single protein molecules, in the environment of choice.[2] The more common AFM imaging capabilities can be coupled with force spectroscopy measurements in order to, for example, first image a membrane and then pull out a protein from it, characterizing the unfolding and the pulling process.[3] In this contribution, we will present the main features of the AFM and force-spectroscopy technique and the results of its use for protein unfolding obtained at our research group. [1] Shevchuk, A.I., G.I. Frolenkov, D. Sánchez, P.S. James, N. Freedman, M.J. Lab, R. Jones, D. Klenerman, and Y.E. Korchev, Imaging Proteins in Membranes of Living Cells by High-Resolution Scanning Ion Conductance Microscopy. Angew Chem Int Ed, 2006. Published Online: 28 Feb 2006. [2] Samorì, B., G. Zuccheri, and P. Baschieri, Protein unfolding and refolding under force: methodologies for nanomechanics. Chemphyschem, 2005. 6(1): p. 29-34. [3] Oesterhelt, F., D. Oesterhelt, M. Pfeiffer, A. Engel, H.E. Gaub, and D.J. Muller, Unfolding pathways of individual bacteriorhodopsins. Science, 2000. 288(5463): p. 143-6.
FIRST INTERNATIONAL WORKSHOP ON EXPRESSION, STRUCTURE AND FUNCTION OF MEMBRANE PROTEINS
G. Zuccheri; F. Valle; M. Sandal; B. Samorì
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/30562
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