Figure 1

Parts of current traces through $\alpha$-hemolysin (at 100 mV) in the presence of MBP, examples of statistical analysis, open pore conductance vs [Gdm-HCl], and events frequency vs MBP and applied voltage. Left (a), without Gdm-HCl the current is constant at 100 pA, MBP cannot enter to the pore; (b) with $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=1.35M$ well above the unfolding transition, the current trace decreases down to 20 pA when a MBP molecule is in the pore. The rms noise of the open pore current does not change at this [Gdm-HCl] value. Right, (c) distribution of blockades time $T_t$; (d) distribution of time intervals $T_i$ between two successive pulses of current (computed over $\simeq 1500$ events). Continuous lines are exponential fits, time constants define the average blockade duration (considering only the exponential tail of the distribution) and average intervals between events. In (a),(b),(c) and (d), $[\mathrm{MBP}]=0.35\mu M$. Bottom, (e) open pore current vs [Gdm-HCl]. The dashed line is a guide to the eye to highlight the slight conductance increase with [Gdm-HCl]. (f) Frequency of events vs [MBP]. The dashed line is a linear fit with a slope of $260\pm 10\mathrm{Hz}\mu M^{-1}$. (g) Frequency of events vs applied voltage on a semilog scale. The dashed line is an exponential fit. The characteristic voltage is $V_0=42\pm 2\mathrm{mV}$.Reuse & Permissions

Figure 2

Part of current traces through $\alpha$-hemolysin pore vs [Gdm-HCl] at $[\mathrm{MBP}]=0.35\mu M$. (a) at $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.8M$, very few short blockades are observed, their average duration is 0.2 ms. (b) at $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.85M$, long blockades of 100 s in average alternate with a series of short ones of 0.2 ms in average. (c) At $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.9M$, the frequency of the short blockades increases (of average duration 0.2 ms). The frequency of the long blockades also increases, their distribution is wide, their mean duration decreases, its value based on the statistical analysis of all events is 40 ms. (d) At $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=1M$, only short blockades (of average duration 0.5 ms) are observed, MBP molecules are almost completely unfolded. The frequency of short events does not further increase.Reuse & Permissions

Figure 3

The nanopore technique detects the unfolding of proteins and the existence of long transient glassy conformations. Here $[\mathrm{MBP}]=0.35\mu M$. (a) Frequency of short blockades vs [Gdm-HCl]. The dashed line is a fit using a Fermi-Dirac sigmoid function. No blockade is observed below $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.8M$. At the unfolding transition, events frequency increases sharply with [Gdm-HCl]. (b) Integrated distribution of the blockade duration, i.e., number of blockade events with duration less than a given time $t$ at $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.9M$. This time distribution is not monoexponential. The line is a fit using a two exponentials distribution. The mean duration of long time blockades is determined from this analysis when statistics is large enough (case of $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.9$ and $1M$) or directly by averaging durations of long blockades when events are sparse (case of $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=0.85$ and $0.875M$). (c) Vogel-Tammann-Fulcher representation of long blockades duration $\tau$ vs $1/(C-C_g)$, with $C=[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]$ and $C_g=0.8M$. The continuous line is the best fit of the form $\tau ={\tau }_0\exp \mathbf{(}A/(C-C_g)\mathbf{)}$. The point at $\tau =0.2\mathrm{ms}$ corresponds to the fully denatured state at $[\mathrm{Gdm}\mathrm{\text{−}}\mathrm{HCl}]=1.35M$.Reuse & Permissions