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Proof of dual-topology architecture of Fluc F- channels with monobody blockers.

Stockbridge RB, Koide A, Miller C, Koide S - Nat Commun (2014)

Bottom Line: Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings.Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity.The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA.

ABSTRACT
Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in their quaternary architecture: they are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here, we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion manoeuvre, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

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Bimolecular kinetics of monobody block(a) Single Bpe recordings with indicated concentrations of monobody L3 in cis chamber, with conditions as in Fig 2. (b) Cumulative distributions of blocked and open dwell times (n=20 for each) for a single Bpe channel in the presence of 30 nM L3 monobody. Solid lines show single-exponential fits. (c) Dependence of blocked (open points) and open (closed points) time constants on monobody concentration [Mb], for L3 block of Bpe. Each point represents mean ± s.e.m. time constant from 3-6 separate bilayers. Solid lines show fits to Eq. 1a and Eq. 1b for open and blocked intervals, respectively, with τB= 41 s (koff=.025 s-1), τO=77 s (kon = 4.3 × 105 s-1M-1), KD = 58 nM.
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Figure 3: Bimolecular kinetics of monobody block(a) Single Bpe recordings with indicated concentrations of monobody L3 in cis chamber, with conditions as in Fig 2. (b) Cumulative distributions of blocked and open dwell times (n=20 for each) for a single Bpe channel in the presence of 30 nM L3 monobody. Solid lines show single-exponential fits. (c) Dependence of blocked (open points) and open (closed points) time constants on monobody concentration [Mb], for L3 block of Bpe. Each point represents mean ± s.e.m. time constant from 3-6 separate bilayers. Solid lines show fits to Eq. 1a and Eq. 1b for open and blocked intervals, respectively, with τB= 41 s (koff=.025 s-1), τO=77 s (kon = 4.3 × 105 s-1M-1), KD = 58 nM.

Mentions: We chose one of the channel-monobody pairs, Bpe // Mb(Bpe_L3), for a quantitative examination of the blocking process. Single-channel recordings (Fig 3A) in the presence of this monobody (henceforth abbreviated ‘L3’) show that both blocked and open intervals are exponentially distributed (Fig 3B), with time constants τB and τo, respectively. Open times shorten and block times remain constant as monobody concentration increases (Fig 3C), in quantitative agreement with expectations of a bimolecular blocking scheme with rate constants of blocker association (kon) and dissociation (koff):IkonOpen channel+Mb<===>Blocked channelkoff(1a)1/τo=kon[Mb](1b)1/τB=koff


Proof of dual-topology architecture of Fluc F- channels with monobody blockers.

Stockbridge RB, Koide A, Miller C, Koide S - Nat Commun (2014)

Bimolecular kinetics of monobody block(a) Single Bpe recordings with indicated concentrations of monobody L3 in cis chamber, with conditions as in Fig 2. (b) Cumulative distributions of blocked and open dwell times (n=20 for each) for a single Bpe channel in the presence of 30 nM L3 monobody. Solid lines show single-exponential fits. (c) Dependence of blocked (open points) and open (closed points) time constants on monobody concentration [Mb], for L3 block of Bpe. Each point represents mean ± s.e.m. time constant from 3-6 separate bilayers. Solid lines show fits to Eq. 1a and Eq. 1b for open and blocked intervals, respectively, with τB= 41 s (koff=.025 s-1), τO=77 s (kon = 4.3 × 105 s-1M-1), KD = 58 nM.
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Related In: Results  -  Collection

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Figure 3: Bimolecular kinetics of monobody block(a) Single Bpe recordings with indicated concentrations of monobody L3 in cis chamber, with conditions as in Fig 2. (b) Cumulative distributions of blocked and open dwell times (n=20 for each) for a single Bpe channel in the presence of 30 nM L3 monobody. Solid lines show single-exponential fits. (c) Dependence of blocked (open points) and open (closed points) time constants on monobody concentration [Mb], for L3 block of Bpe. Each point represents mean ± s.e.m. time constant from 3-6 separate bilayers. Solid lines show fits to Eq. 1a and Eq. 1b for open and blocked intervals, respectively, with τB= 41 s (koff=.025 s-1), τO=77 s (kon = 4.3 × 105 s-1M-1), KD = 58 nM.
Mentions: We chose one of the channel-monobody pairs, Bpe // Mb(Bpe_L3), for a quantitative examination of the blocking process. Single-channel recordings (Fig 3A) in the presence of this monobody (henceforth abbreviated ‘L3’) show that both blocked and open intervals are exponentially distributed (Fig 3B), with time constants τB and τo, respectively. Open times shorten and block times remain constant as monobody concentration increases (Fig 3C), in quantitative agreement with expectations of a bimolecular blocking scheme with rate constants of blocker association (kon) and dissociation (koff):IkonOpen channel+Mb<===>Blocked channelkoff(1a)1/τo=kon[Mb](1b)1/τB=koff

Bottom Line: Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings.Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity.The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, USA.

ABSTRACT
Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in their quaternary architecture: they are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here, we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion manoeuvre, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

Show MeSH
Related in: MedlinePlus