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A kinetic analysis of protein transport through the anthrax toxin channel.

Basilio D, Kienker PK, Briggs SW, Finkelstein A - J. Gen. Physiol. (2011)

Bottom Line: As expected, the translocation rate is slower with more than one LF(N) bound.We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field.The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dab2043@-med.cornell.edu

ABSTRACT
Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

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Illustration of all the possible ways that the seven binding sites on the (PA63)7 channel can be occupied by LFN. The ring of seven gray circles represents the (PA63)7 channel, and the black oval represents LFN. Note that when LFN is bound to the channel, it occupies two adjacent subunits. kon is the rate constant for binding of LFN to two subunits, and koff is the dissociation rate. (kon is directly proportional to the concentration of LFN.) The arrows indicate the binding and unbinding transitions between each of the states. [0], [1], [2], and [3] are the fractions of channels occupied by zero, one, two, and three LFNs, respectively.
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fig3: Illustration of all the possible ways that the seven binding sites on the (PA63)7 channel can be occupied by LFN. The ring of seven gray circles represents the (PA63)7 channel, and the black oval represents LFN. Note that when LFN is bound to the channel, it occupies two adjacent subunits. kon is the rate constant for binding of LFN to two subunits, and koff is the dissociation rate. (kon is directly proportional to the concentration of LFN.) The arrows indicate the binding and unbinding transitions between each of the states. [0], [1], [2], and [3] are the fractions of channels occupied by zero, one, two, and three LFNs, respectively.

Mentions: Although there are seven potential binding sites for LFN, maximally only three can be occupied (Fig. 1; Cunningham et al., 2002; Mogridge et al., 2002; Pimental et al., 2004). Let kon (which depends on the concentration of LFN) be the on-rate constant for a given site, and let koff be the off-rate constant. We assume, as shown for the whole LF (Elliott et al., 2000; Neumeyer et al., 2006), that these rate constants are independent of the number of sites occupied; therefore, at equilibrium the transitions between the different occupied states (Fig. 3) are characterized by the following set of equations:7kon[0]=koff[1],4kon[1]=2koff[2],1.5kon[2]=3koff[3],and[0]+[1]+[2]+[3]=1,where [0], [1], [2], and [3] are the fractions of channels occupied by zero, one, two, and three LFNs, respectively. Solving these equations, we obtain(1)[0]= 1/z,[1]= 7K/z,[2]=14K2/z, and[3]= 7K3/z,whereK=kon/koffandz=1+7K+14K2+7K3.Given [0], which is the experimentally observed quantity, we can solve for K and thus determine [1], [2], and [3]. Plots of [1], [2], and [3] as a function of [0] are shown in Fig. 4.


A kinetic analysis of protein transport through the anthrax toxin channel.

Basilio D, Kienker PK, Briggs SW, Finkelstein A - J. Gen. Physiol. (2011)

Illustration of all the possible ways that the seven binding sites on the (PA63)7 channel can be occupied by LFN. The ring of seven gray circles represents the (PA63)7 channel, and the black oval represents LFN. Note that when LFN is bound to the channel, it occupies two adjacent subunits. kon is the rate constant for binding of LFN to two subunits, and koff is the dissociation rate. (kon is directly proportional to the concentration of LFN.) The arrows indicate the binding and unbinding transitions between each of the states. [0], [1], [2], and [3] are the fractions of channels occupied by zero, one, two, and three LFNs, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105512&req=5

fig3: Illustration of all the possible ways that the seven binding sites on the (PA63)7 channel can be occupied by LFN. The ring of seven gray circles represents the (PA63)7 channel, and the black oval represents LFN. Note that when LFN is bound to the channel, it occupies two adjacent subunits. kon is the rate constant for binding of LFN to two subunits, and koff is the dissociation rate. (kon is directly proportional to the concentration of LFN.) The arrows indicate the binding and unbinding transitions between each of the states. [0], [1], [2], and [3] are the fractions of channels occupied by zero, one, two, and three LFNs, respectively.
Mentions: Although there are seven potential binding sites for LFN, maximally only three can be occupied (Fig. 1; Cunningham et al., 2002; Mogridge et al., 2002; Pimental et al., 2004). Let kon (which depends on the concentration of LFN) be the on-rate constant for a given site, and let koff be the off-rate constant. We assume, as shown for the whole LF (Elliott et al., 2000; Neumeyer et al., 2006), that these rate constants are independent of the number of sites occupied; therefore, at equilibrium the transitions between the different occupied states (Fig. 3) are characterized by the following set of equations:7kon[0]=koff[1],4kon[1]=2koff[2],1.5kon[2]=3koff[3],and[0]+[1]+[2]+[3]=1,where [0], [1], [2], and [3] are the fractions of channels occupied by zero, one, two, and three LFNs, respectively. Solving these equations, we obtain(1)[0]= 1/z,[1]= 7K/z,[2]=14K2/z, and[3]= 7K3/z,whereK=kon/koffandz=1+7K+14K2+7K3.Given [0], which is the experimentally observed quantity, we can solve for K and thus determine [1], [2], and [3]. Plots of [1], [2], and [3] as a function of [0] are shown in Fig. 4.

Bottom Line: As expected, the translocation rate is slower with more than one LF(N) bound.We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field.The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. dab2043@-med.cornell.edu

ABSTRACT
Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Show MeSH
Related in: MedlinePlus