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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.


a Room-temperature CW-EPR of MscL labeled with EPR spin label at position number 22nd (right panel), 24th (middle panel), and 28th (right panel) showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (10:1 molar ratio, spin label/subunit). Traces in red were obtained from under-labeled channels (1:10 molar ratio, spin label/subunit). b Inverse values of the interaction parameter (Ω−1) as a function of the time of irradiation
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Fig5: a Room-temperature CW-EPR of MscL labeled with EPR spin label at position number 22nd (right panel), 24th (middle panel), and 28th (right panel) showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (10:1 molar ratio, spin label/subunit). Traces in red were obtained from under-labeled channels (1:10 molar ratio, spin label/subunit). b Inverse values of the interaction parameter (Ω−1) as a function of the time of irradiation

Mentions: Figure 5a shows the different degrees of spectral broadening due to channel opening for these residues. We quantified the line broadening by using the inverse values of the interaction parameter, Ω−1 again. Figure 5b shows the inverse values of the interaction parameter as a function of time of irradiation for the selected residues I24C and A28C. We observed a similar Ω−1 value for all the residues after 90 s of irradiation, although the trend of the increase was different for each residue. At the end of 180 s of irradiation, the biggest increase in Ω−1 was observed for G22C, followed by A28C and I24C, respectively. Based on our results, we conclude that when there are three light switches present per pentamer, among the three residues we selected, G22C position is subjected to the biggest change during channel gating.Fig. 5


Study of light-induced MscL gating by EPR spectroscopy.

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

a Room-temperature CW-EPR of MscL labeled with EPR spin label at position number 22nd (right panel), 24th (middle panel), and 28th (right panel) showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (10:1 molar ratio, spin label/subunit). Traces in red were obtained from under-labeled channels (1:10 molar ratio, spin label/subunit). b Inverse values of the interaction parameter (Ω−1) as a function of the time of irradiation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: a Room-temperature CW-EPR of MscL labeled with EPR spin label at position number 22nd (right panel), 24th (middle panel), and 28th (right panel) showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (10:1 molar ratio, spin label/subunit). Traces in red were obtained from under-labeled channels (1:10 molar ratio, spin label/subunit). b Inverse values of the interaction parameter (Ω−1) as a function of the time of irradiation
Mentions: Figure 5a shows the different degrees of spectral broadening due to channel opening for these residues. We quantified the line broadening by using the inverse values of the interaction parameter, Ω−1 again. Figure 5b shows the inverse values of the interaction parameter as a function of time of irradiation for the selected residues I24C and A28C. We observed a similar Ω−1 value for all the residues after 90 s of irradiation, although the trend of the increase was different for each residue. At the end of 180 s of irradiation, the biggest increase in Ω−1 was observed for G22C, followed by A28C and I24C, respectively. Based on our results, we conclude that when there are three light switches present per pentamer, among the three residues we selected, G22C position is subjected to the biggest change during channel gating.Fig. 5

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.