This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.125807v1 94/10/3945    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Koehorst, R. B. M. Articles by Hemminga, M. A. Search for Related Content PubMed PubMed Citation Articles by Koehorst, R. B. M. Articles by Hemminga, M. A.

Site-Directed Fluorescence Labeling of a Membrane Protein with BADAN: Probing Protein Topology and Local Environment

Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands

Correspondence: Address reprint requests to Marcus A. Hemminga, Laboratory of Biophysics, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands. Office: Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. Tel.: 31-317-482044; Fax: 31-317-482725; E-mail: marcus.hemminga{at}wur.nl; web site: http://ntmf.mf.wau.nl/hemminga/.

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly -helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.