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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.


MscL treated with different denaturants ran on blue native gel. A portion of pure G22C MscL was treated with a SDS (final concentration 0.6 % w/v), b urea (final concentration 2 and 6 M), c trifluoroethanol (TFE) (final concentration 2 and 4 % w/v), and d guanidinium hydrochloride (final concentration 2 and 6 M). The black and white arrows show the pentameric and smaller oligomeric forms of MscL, respectively
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Fig3: MscL treated with different denaturants ran on blue native gel. A portion of pure G22C MscL was treated with a SDS (final concentration 0.6 % w/v), b urea (final concentration 2 and 6 M), c trifluoroethanol (TFE) (final concentration 2 and 4 % w/v), and d guanidinium hydrochloride (final concentration 2 and 6 M). The black and white arrows show the pentameric and smaller oligomeric forms of MscL, respectively

Mentions: In order to dissociate MscL pentamers into monomers, we first screened various denaturants including urea, guanidinium hydrochloride, trifluoroethanol (TFE) (Fig. 3a–d), and various detergents such as Triton X-100, C12E9, foscholine 10, nonyl-glucoside, decyl-glucoside, cymal 3, CHAPS, CHAPSO, and SDS. None of the indicated compounds could dissociate the pentamers of MscL into monomers (Fig. 3b–d, black arrow), except for SDS (Fig. 3a, white arrows). However, the removal of SDS from the channel was problematic in the further steps.Fig. 3


Study of light-induced MscL gating by EPR spectroscopy.

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

MscL treated with different denaturants ran on blue native gel. A portion of pure G22C MscL was treated with a SDS (final concentration 0.6 % w/v), b urea (final concentration 2 and 6 M), c trifluoroethanol (TFE) (final concentration 2 and 4 % w/v), and d guanidinium hydrochloride (final concentration 2 and 6 M). The black and white arrows show the pentameric and smaller oligomeric forms of MscL, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: MscL treated with different denaturants ran on blue native gel. A portion of pure G22C MscL was treated with a SDS (final concentration 0.6 % w/v), b urea (final concentration 2 and 6 M), c trifluoroethanol (TFE) (final concentration 2 and 4 % w/v), and d guanidinium hydrochloride (final concentration 2 and 6 M). The black and white arrows show the pentameric and smaller oligomeric forms of MscL, respectively
Mentions: In order to dissociate MscL pentamers into monomers, we first screened various denaturants including urea, guanidinium hydrochloride, trifluoroethanol (TFE) (Fig. 3a–d), and various detergents such as Triton X-100, C12E9, foscholine 10, nonyl-glucoside, decyl-glucoside, cymal 3, CHAPS, CHAPSO, and SDS. None of the indicated compounds could dissociate the pentamers of MscL into monomers (Fig. 3b–d, black arrow), except for SDS (Fig. 3a, white arrows). However, the removal of SDS from the channel was problematic in the further steps.Fig. 3

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.