The Effect of Temperature on Mechanical Resistance of the Native and Intermediate States of I27

Abstract

We investigated the effect of temperature on the mechanical unfolding of I27 from human cardiac titin, employing a custom-built temperature control device for single-molecule atomic force microscopy measurement. A sawtooth pattern was observed in the force curves where each force peak reports on the unfolding of an I27 domain. In early unfolding events, we observed a "hump-like" deviation due to the detachment of beta-strand A from each I27 domain. The force at which the humps appear was approximately 130 pN and showed no temperature dependence, at least in the temperature range of 2 degrees C-30 degrees C. The hump structure was successfully analyzed with a two-state worm-like chain model, and the Gibbs free energy difference of the detachment reaction was estimated to be 11.6 +/- 0.58 kcal/mol and found to be temperature independent. By contrast, upon lowering the temperature, the mean unfolding force from the partly unfolded intermediate state was found to markedly increase and the unfolding force distribution to broaden significantly, suggesting that the distance (x(u)) between the folded and transition states in the energy landscape along the pulling direction is decreased. These results suggest that the local structure of beta-strand A are stabilized by topologically simple local hydrogen-bond network and that the temperature does not affect the detachment reaction thermodynamically and kinetically, whereas the interaction between the beta-strands A' and G, which is a critical region for its mechanical stability, is strongly dependent on the temperature.

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

“…By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range 5-45°C, we measure a value for the activation barrier of ⌬G ‡ ϭ 71 Ϯ 5 kJ/mol and an exponential prefactor ‡ ϳ4 ϫ 10 9 s Ϫ1 . Contrary to the results reported for filamin (25), and similar to the case of the I27 protein (26), the major effect of temperature is to significantly lower the height of the energy barrier, whereas the distance to the transition state increases only slightly within the range of temperatures probed in our experiments. The measured value for the exponential prefactor for mechanical unfolding, ‡ , on the nanosecond timescale, places an upper limit to the diffusive mean first passage time for unfolding.…”

“…These studies have provided valuable information regard-ing their nanomechanical properties, which are intimately related to the structural topology. Previous single molecule mechanical studies have investigated the effect of temperature on the nanomechanical properties of proteins (23)(24)(25)(26)(27)(28). Qualitatively, these works reported an expected decrease in the mechanical stability of the protein as the temperature was increased.…”

Computational investigation of the effect of thermal perturbation on the mechanical unfolding of titin I27

“…Traditionally, chemical denaturants such as guanidinium chloride and thermal perturbation along with spectroscopic methods have been used to study protein folding [9]. Recent studies have shown that it is important to combine single-molecule experiments with either chemical denaturants or with thermal perturbations to study folding processes [10][11][12][13][14]. They proposed that such combinations of multiple techniques along with comparisons of results from multiple probes are useful for validating the protocol used [12].…”

“…The protein was selected based on recent experimental studies of this protein [11,13], and the validity of the end-to-end distance as the order parameter for the unfolding of this protein. Botello et al showed that the free-energy barrier during the mechanical unfolding of I27 decreases linearly with respect to increasing temperature and the concentration of the chemical denaturant, guanidinium chloride [11].…”

“…Botello et al showed that the free-energy barrier during the mechanical unfolding of I27 decreases linearly with respect to increasing temperature and the concentration of the chemical denaturant, guanidinium chloride [11]. A detailed study by Taniguchi et al illustrated the effect of temperature on the mechanical stress of the native and intermediate states of I27 [13]. They hypothesized that the energy landscape does not alter with respect to temperature until the folded state reaches the intermediate state, although it changes substantially beyond that.…”