科研之友
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摘要
By using single-molecule Forster resonance energy transfer (smFRET), we have studied RNase H in the presence of the denaturant guanidinium chloride to explore its well-known folding intermediate. FRET efficiency histograms were determined from experiments on freely diffusing proteins at pH 3. Even with excellent data statistics, a folding intermediate is not obvious from the FRET efficiency histograms, owing to the broad and overlapping distributions associated with each of the three macroscopic (folded, intermediate, unfolded) states. We developed a global fitting procedure for the smFRET data based on a thermodynamic model that assumes the free energies of the three states to vary linearly with denaturant activity. In addition, we included tryptophan emission data measured on bulk samples, which place significant constraints on the relative populations of the three states. The analysis yields free energy differences between the states and, thus, fractional populations over the entire range of denaturant concentration.
- 出版日期2012-3-2
