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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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The half-time of translocation as a function of voltage. The values for WT LFN (circles) and His6-LFN (triangles) are from the experiments described in Fig. 9. Note that at every voltage, the rate of translocation of His6-LFN is slower than that of WT LFN but that the rates tend to converge as the voltage increases. Thus, His6-LFNt1/2/WT LFNt1/2 = 3.3, 2.3, 1.6, and 1.1 for V = 45, 50, 55, and 60 mV, respectively. The fitted curves were calculated from the drift-diffusion model as described in the Results section.
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fig11: The half-time of translocation as a function of voltage. The values for WT LFN (circles) and His6-LFN (triangles) are from the experiments described in Fig. 9. Note that at every voltage, the rate of translocation of His6-LFN is slower than that of WT LFN but that the rates tend to converge as the voltage increases. Thus, His6-LFNt1/2/WT LFNt1/2 = 3.3, 2.3, 1.6, and 1.1 for V = 45, 50, 55, and 60 mV, respectively. The fitted curves were calculated from the drift-diffusion model as described in the Results section.

Mentions: We have generally observed that the translocation of His6-LFN is significantly slower than that of WT LFN (which has the His6 tag removed). (Indeed, we took advantage of this property to facilitate the measurement of the lag time in the single-channel translocation experiments.) To confirm this, we compared the translocation kinetics of WT LFN and His6-LFN on the same membrane using our standard ∼95% block condition. And indeed, at a given voltage, t1/2 for His6-LFN is greater than that for WT LFN (see Fig. 11). Fig. 9 shows the normalized conductance plotted against normalized time (t/t1/2) for WT LFN (A−D) and His6-LFN (E−H) over a range of voltages. The superimposed blue curves are W(Ω, t′) for the best fitted values of Ω. We see that the translocation kinetics of WT LFN are more sigmoidal (i.e., have more positive Ω) than those of His6-LFN.


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

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

The half-time of translocation as a function of voltage. The values for WT LFN (circles) and His6-LFN (triangles) are from the experiments described in Fig. 9. Note that at every voltage, the rate of translocation of His6-LFN is slower than that of WT LFN but that the rates tend to converge as the voltage increases. Thus, His6-LFNt1/2/WT LFNt1/2 = 3.3, 2.3, 1.6, and 1.1 for V = 45, 50, 55, and 60 mV, respectively. The fitted curves were calculated from the drift-diffusion model as described in the Results section.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig11: The half-time of translocation as a function of voltage. The values for WT LFN (circles) and His6-LFN (triangles) are from the experiments described in Fig. 9. Note that at every voltage, the rate of translocation of His6-LFN is slower than that of WT LFN but that the rates tend to converge as the voltage increases. Thus, His6-LFNt1/2/WT LFNt1/2 = 3.3, 2.3, 1.6, and 1.1 for V = 45, 50, 55, and 60 mV, respectively. The fitted curves were calculated from the drift-diffusion model as described in the Results section.
Mentions: We have generally observed that the translocation of His6-LFN is significantly slower than that of WT LFN (which has the His6 tag removed). (Indeed, we took advantage of this property to facilitate the measurement of the lag time in the single-channel translocation experiments.) To confirm this, we compared the translocation kinetics of WT LFN and His6-LFN on the same membrane using our standard ∼95% block condition. And indeed, at a given voltage, t1/2 for His6-LFN is greater than that for WT LFN (see Fig. 11). Fig. 9 shows the normalized conductance plotted against normalized time (t/t1/2) for WT LFN (A−D) and His6-LFN (E−H) over a range of voltages. The superimposed blue curves are W(Ω, t′) for the best fitted values of Ω. We see that the translocation kinetics of WT LFN are more sigmoidal (i.e., have more positive Ω) than those of His6-LFN.

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