Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment.
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 alpha-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.
Koehorst
Rob B M
RB
Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands.
Spruijt
Ruud B
RB
Hemminga
Marcus A
MA
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D003198:Computer Simulation; D003461:Crystallography / Q000379:methods*; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods*
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D008051:Lipid Bilayers; D008565:Membrane Proteins; D015081:2-Naphthylamine