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Study of light-induced MscL gating by EPR spectroscopy.

Yilmaz D, Dimitrova AI, Walko M, Kocer A - Eur. Biophys. J. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.

ABSTRACT
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.

No MeSH data available.


Method of generating heteropentamers of MscL with defined number of light switches and EPR spin labels
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Fig2: Method of generating heteropentamers of MscL with defined number of light switches and EPR spin labels

Mentions: Here, we present an approach that enables activation of MscL into initial sub-open states in a well-controlled manner by using light. We labeled MscL in its hydrophobic pore region on an engineered cysteine residue at the G22 position using a cysteine-selective alkylating reagent. The reagent is composed of an iodoacetate bearing the photocleavable protecting group 6-nitroveratryl alcohol (Fig. 2, Kocer et al. 2005). This compound is sensitive to long-wavelength ultraviolet. Upon illumination at λ > 300 nm, the photolysis of the protective group leaves negatively charged cysteine-bound acetates at the pore of MscL, which activated the channel with hydrophobic gating mechanism (Birkner et al. 2012). During the channel opening, we monitor the resulting structural changes on MscL by employing spin-label EPR spectroscopy. We believe that controlled activation of MscL into defined sub-open states from the initiation of its pore opening would help understanding the initiation of mechanosensation process at the molecular level. Furthermore, the method also offers labeling the desired number of monomers within a pentamer. Therefore, it can also be used to avoid the dipolar spin and spin–exchange interactions.Fig. 2


Study of light-induced MscL gating by EPR spectroscopy.

Yilmaz D, Dimitrova AI, Walko M, Kocer A - Eur. Biophys. J. (2015)

Method of generating heteropentamers of MscL with defined number of light switches and EPR spin labels
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562997&req=5

Fig2: Method of generating heteropentamers of MscL with defined number of light switches and EPR spin labels
Mentions: Here, we present an approach that enables activation of MscL into initial sub-open states in a well-controlled manner by using light. We labeled MscL in its hydrophobic pore region on an engineered cysteine residue at the G22 position using a cysteine-selective alkylating reagent. The reagent is composed of an iodoacetate bearing the photocleavable protecting group 6-nitroveratryl alcohol (Fig. 2, Kocer et al. 2005). This compound is sensitive to long-wavelength ultraviolet. Upon illumination at λ > 300 nm, the photolysis of the protective group leaves negatively charged cysteine-bound acetates at the pore of MscL, which activated the channel with hydrophobic gating mechanism (Birkner et al. 2012). During the channel opening, we monitor the resulting structural changes on MscL by employing spin-label EPR spectroscopy. We believe that controlled activation of MscL into defined sub-open states from the initiation of its pore opening would help understanding the initiation of mechanosensation process at the molecular level. Furthermore, the method also offers labeling the desired number of monomers within a pentamer. Therefore, it can also be used to avoid the dipolar spin and spin–exchange interactions.Fig. 2

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.

ABSTRACT
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.

No MeSH data available.