Study of light-induced MscL gating by EPR spectroscopy.
A number of techniques developed to investigate protein structure and function depend on chemically modifying and/or labeling of proteins. However, in the case of homooligomeric proteins, the presence of multiple identical subunits obstructs the introduction of residue-specific labels to only one or several subunits, selectively. Here, in order to study the initial conformational changes of a homopentameric mechanosensitive ion channel during its gating, we developed a method for labeling a defined number of subunits of the channel with two different cysteine-specific compounds simultaneously. The first one is a light-sensitive channel activator that determines the degree of openness of the ion channel upon irradiation. The second one is a spin label, containing an unpaired electron, which allows following the resulting structural changes upon channel gating by electron paramagnetic resonance spectroscopy. With this method, we could open MscL into different sub-open states. As the number of light switches per channel increased, the intersubunit spin-spin interactions became less, indicating changes in intersubunit proximities and opening of the channel. The ability of controlled activation of MscL into different open states with a noninvasive trigger and following the resulting conformational changes by spectroscopy will pave the way for detailed spectroscopic studies in the area of mechanosensation.
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Duygu
D
Yilmaz
Anna I
AI
Dimitrova
Martin
M
Walko
Armagan
A
Kocer
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D008027:Light; D040542:Mechanotransduction, Cellular; D008969:Molecular Sequence Data
D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli