Staff Publications

Staff Publications

  • external user (warningwarning)
  • Log in as
  • language uk
  • About

    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

    We have a manual that explains all the features 

Record number 352236
Title Distance constraints from site-directed spectroscopy as a tool to study membrane protein structure
Author(s) Vos, W.L.
Source Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga. - [S.l.] : S.n. - ISBN 9789085046257 - 106
Department(s) Biophysics
Publication type Dissertation, internally prepared
Publication year 2007
Keyword(s) oppervlakte-eiwitten - moleculaire structuur - spectroscopie - surface proteins - molecular conformation - spectroscopy
Categories Membranes
Abstract Membrane proteins are involved in nearly every process in the living cell. Their scientific importance cannot be overstated, and they account for nearly 60% of all prescribed drugs. Despite being an abundant and important class of proteins, high-resolution structural data on membrane proteins are relatively scarce. X-ray diffraction and NMR spectroscopy are routinely applied nowadays for the determination of structures of water-soluble proteins. However, for membrane proteins that require an amphipathic environment, there is not yet a well-defined strategy for obtaining the structure. For this reason, techniques based on site-directed labeling are being developed to study membrane proteins in their natural environment. In this work, we use two techniques based on the dipole-dipole interaction between two labels, electron spin resonance (ESR) and fluorescence (or Förster) resonance energy transfer (FRET) to obtain low-resolution (0.3-3 nm) distance information on the structure of membrane peptides. FRET is used to study the conformation of a reference membrane protein, i.e. M13 major coat protein, in fully hydrated vesicles. The FRET-derived distance constraints are used to refine the set of high-resolution structures that is available in the protein databank. We show that the coat protein adopts an extended conformation that is not very different from the conformation in the phage particle. In a separate part of this work, we use the FRET approach to monitor the conformation of the coat protein under conditions of hydrophobic mismatch. Although it was suggested that transmembrane protein domains can adapt their backbone conformation to different conditions of hydrophobic stress and that M13 coat protein is a flexible protein that can adapt to a multitude of environments, we show that the conformation of the coat protein in fact is similar under different conditions of hydrophobic mismatch. A parallel approach, based on ESR spin labeling, is used to study the conformation of a peptide that is derived from the crucial proton translocating domain of vacuolar ATPase. First we present a method to enhance the analysis for the determination of distances between two spin labels based on matrix-assisted laser desorption/ionization - time of flight mass spectrometry. Secondly, we use the data from the ESR experiments to study the structure of the peptide. Based on the combined results from the ESR experiments, molecular dynamics simulations and circular dichroism studies we conclude that the peptide forms a dynamica-helix when bound to SDS micelles. We discuss these findings in the light of the current models for proton translocation in the vacuolar ATPase.
There are no comments yet. You can post the first one!
Post a comment
Please log in to use this service. Login as Wageningen University & Research user or guest user in upper right hand corner of this page.