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High temperature unfolding simulations of the TRPZ1 peptide.

Settanni G, Fersht AR - Biophys. J. (2008)

Bottom Line: But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues.The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments.The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here.

View Article: PubMed Central - PubMed

Affiliation: Centre for Protein Engineering, Cambridge, United Kingdom. gs@mrc-lmb.cam.ac.uk

ABSTRACT
We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 mus with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations. Several unfolding and refolding transition pathways are sampled and analyzed. The unfolding process of the peptide occurs in two steps because of the accumulation of a metastable on-pathway intermediate state stabilized by two native backbone hydrogen bonds assisted by nonnative hydrophobic interactions between the tryptophan side chains. Analysis of the un/folding kinetics and classical commitment probability calculations on the conformations extracted from the transition pathways show that the rate-limiting step for unfolding is the disruption of the ordered native hydrophobic packing (Trp-zip motif) leading from the native to the intermediate state. But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues. The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments. The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here.

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A qualitative picture of the free energy landscape of the TRPZ1 peptide determined on the basis of the transition times between states at different temperatures. TS1 and TS2 indicate the transition states for the N→I and I→D transitions, respectively. The ΔG associated with each barrier has been determined using the formula k = a exp(−ΔG/RT) where R is the gas constant, T is the temperature, k is the measured rate for the transition, and a = bT is the preexponential factor that has been set to (0.5 ns)−1 at 450 K and scaled accordingly at the other temperatures. This value for the preexponential factor has been chosen on the basis of the time measured to observe a complete shift in Pcommit (from 0 to 1 or from 1 to 0) along the trajectory for the I→D transition. Please note that, while the height of the barriers depends on the choice of the preexponential factor, the free energy difference between minima is independent of it. The dashed line indicates that no transition was observed in that case; thus, the corresponding rate is supposed to be smaller than the inverse dwelling time spent by the system in the unproductive state. Error estimates for the free energy differences are usually comprised between 0.2 and 0.5 Kcal/mol, as determined by propagation of the uncertainties on the transition times (Table 3), with the exception of the free energy of TS2 and D at 400 K where the propagated error is 0.9 and 1.0 Kcal/mol, respectively.
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fig4: A qualitative picture of the free energy landscape of the TRPZ1 peptide determined on the basis of the transition times between states at different temperatures. TS1 and TS2 indicate the transition states for the N→I and I→D transitions, respectively. The ΔG associated with each barrier has been determined using the formula k = a exp(−ΔG/RT) where R is the gas constant, T is the temperature, k is the measured rate for the transition, and a = bT is the preexponential factor that has been set to (0.5 ns)−1 at 450 K and scaled accordingly at the other temperatures. This value for the preexponential factor has been chosen on the basis of the time measured to observe a complete shift in Pcommit (from 0 to 1 or from 1 to 0) along the trajectory for the I→D transition. Please note that, while the height of the barriers depends on the choice of the preexponential factor, the free energy difference between minima is independent of it. The dashed line indicates that no transition was observed in that case; thus, the corresponding rate is supposed to be smaller than the inverse dwelling time spent by the system in the unproductive state. Error estimates for the free energy differences are usually comprised between 0.2 and 0.5 Kcal/mol, as determined by propagation of the uncertainties on the transition times (Table 3), with the exception of the free energy of TS2 and D at 400 K where the propagated error is 0.9 and 1.0 Kcal/mol, respectively.

Mentions: Few direct transitions were observed between N and D that had no significant dwelling times spent in I (25% of the total number of transitions with no change of this fraction with temperature), indicating that I is a predominant on-pathway intermediate (Fig. 3). The magnitudes of the transition times between the states as a function of temperature (Table 3) provide much information. At 373 K, only few transitions to I and back to N were observed and the dwelling time in the I state was very small, indicating a poor sampling of this state. At the higher temperatures, transitions to D were also observed. In those cases, the slowest unfolding process is the N→I transition. The completion of the unfolding process by the I→D transition occurs on a shorter timescale. As expected in high temperature unfolding simulations, statistics for the folding process is scarcer than for the unfolding process. In the case of temperatures above 435 K, the statistics is sufficient to draw a qualitative picture of the underlying free energy landscape of the peptide (Fig. 4). As expected in nonequilibrium unfolding simulations, the measured population of each state (Table 2) does not match information on the relative free energy as measured by kinetic rates (Fig. 4). The N state is overpopulated because all the simulations were started from that state. However, the I state is correctly found with a population smaller than D.


High temperature unfolding simulations of the TRPZ1 peptide.

Settanni G, Fersht AR - Biophys. J. (2008)

A qualitative picture of the free energy landscape of the TRPZ1 peptide determined on the basis of the transition times between states at different temperatures. TS1 and TS2 indicate the transition states for the N→I and I→D transitions, respectively. The ΔG associated with each barrier has been determined using the formula k = a exp(−ΔG/RT) where R is the gas constant, T is the temperature, k is the measured rate for the transition, and a = bT is the preexponential factor that has been set to (0.5 ns)−1 at 450 K and scaled accordingly at the other temperatures. This value for the preexponential factor has been chosen on the basis of the time measured to observe a complete shift in Pcommit (from 0 to 1 or from 1 to 0) along the trajectory for the I→D transition. Please note that, while the height of the barriers depends on the choice of the preexponential factor, the free energy difference between minima is independent of it. The dashed line indicates that no transition was observed in that case; thus, the corresponding rate is supposed to be smaller than the inverse dwelling time spent by the system in the unproductive state. Error estimates for the free energy differences are usually comprised between 0.2 and 0.5 Kcal/mol, as determined by propagation of the uncertainties on the transition times (Table 3), with the exception of the free energy of TS2 and D at 400 K where the propagated error is 0.9 and 1.0 Kcal/mol, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2480669&req=5

fig4: A qualitative picture of the free energy landscape of the TRPZ1 peptide determined on the basis of the transition times between states at different temperatures. TS1 and TS2 indicate the transition states for the N→I and I→D transitions, respectively. The ΔG associated with each barrier has been determined using the formula k = a exp(−ΔG/RT) where R is the gas constant, T is the temperature, k is the measured rate for the transition, and a = bT is the preexponential factor that has been set to (0.5 ns)−1 at 450 K and scaled accordingly at the other temperatures. This value for the preexponential factor has been chosen on the basis of the time measured to observe a complete shift in Pcommit (from 0 to 1 or from 1 to 0) along the trajectory for the I→D transition. Please note that, while the height of the barriers depends on the choice of the preexponential factor, the free energy difference between minima is independent of it. The dashed line indicates that no transition was observed in that case; thus, the corresponding rate is supposed to be smaller than the inverse dwelling time spent by the system in the unproductive state. Error estimates for the free energy differences are usually comprised between 0.2 and 0.5 Kcal/mol, as determined by propagation of the uncertainties on the transition times (Table 3), with the exception of the free energy of TS2 and D at 400 K where the propagated error is 0.9 and 1.0 Kcal/mol, respectively.
Mentions: Few direct transitions were observed between N and D that had no significant dwelling times spent in I (25% of the total number of transitions with no change of this fraction with temperature), indicating that I is a predominant on-pathway intermediate (Fig. 3). The magnitudes of the transition times between the states as a function of temperature (Table 3) provide much information. At 373 K, only few transitions to I and back to N were observed and the dwelling time in the I state was very small, indicating a poor sampling of this state. At the higher temperatures, transitions to D were also observed. In those cases, the slowest unfolding process is the N→I transition. The completion of the unfolding process by the I→D transition occurs on a shorter timescale. As expected in high temperature unfolding simulations, statistics for the folding process is scarcer than for the unfolding process. In the case of temperatures above 435 K, the statistics is sufficient to draw a qualitative picture of the underlying free energy landscape of the peptide (Fig. 4). As expected in nonequilibrium unfolding simulations, the measured population of each state (Table 2) does not match information on the relative free energy as measured by kinetic rates (Fig. 4). The N state is overpopulated because all the simulations were started from that state. However, the I state is correctly found with a population smaller than D.

Bottom Line: But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues.The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments.The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here.

View Article: PubMed Central - PubMed

Affiliation: Centre for Protein Engineering, Cambridge, United Kingdom. gs@mrc-lmb.cam.ac.uk

ABSTRACT
We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 mus with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations. Several unfolding and refolding transition pathways are sampled and analyzed. The unfolding process of the peptide occurs in two steps because of the accumulation of a metastable on-pathway intermediate state stabilized by two native backbone hydrogen bonds assisted by nonnative hydrophobic interactions between the tryptophan side chains. Analysis of the un/folding kinetics and classical commitment probability calculations on the conformations extracted from the transition pathways show that the rate-limiting step for unfolding is the disruption of the ordered native hydrophobic packing (Trp-zip motif) leading from the native to the intermediate state. But, the speed of the folding process is mainly determined by the transition from the completely unfolded state to the intermediate and specifically by the closure of the hairpin loop driven by formation of two native backbone hydrogen bonds and hydrophobic contacts between tryptophan residues. The temperature dependence of the unfolding time provides an estimate of the unfolding activation enthalpy that is in agreement with experiments. The unfolding time extrapolated to room temperature is in agreement with the experimental data as well, thus providing a further validation to the analysis reported here.

Show MeSH
Related in: MedlinePlus