Sub-Millisecond Unfolding Kinetic Spectra Reveals Intermediate Transitions

Abstract

The formation of stable partially folded structures on the folding pathway of a protein plays an important role for its correct function. Intermediate states mediate protein (sub-) domain folds to reach the most stable native conformational state. Serious diseases can be triggered if proteins are incorrectly folded stabilizing misfolded conformational states. Currently, no single biophysical tool exist that could provide the overall map all conformational transitions during fondling or unfolding kinetics. Here, we used the model enzyme lysozyme from the phage T4 (T4L), a simple two-subdomain protein, from which is known from prior NMR and stopped-flow experiments that it unfolds via at least two intermediate states. To complete the kinetic gap, we created a library of double mutants of the cysteine-free pseudo-wild type by inserting an unnatural amino acid and a cysteine mutation. We proceeded to site-specifically labeled them using orthogonal chemistry with a Forster resonance energy transfer (FRET) dye pair to build up a network of distance restrains spanning the enzyme. The unfolding process was monitored using traditional ensemble methods like CD- spectroscopy and fluorescence spectroscopic methods (ensemble time correlated single photon counting, single molecule fluorescence spectroscopy, (filtered) fluorescence correlation spectroscopy). Single molecule high precision FRET, with its high temporal and spatial resolution, covering over seven orders of magnitude on time, enables us to determine equilibrium transition rates between conformational states in denatured conditions. By using a priori knowledge from earlier studies, we are able to assign the formerly unknown rates to the folding and unfolding intermediate formation steps. The previously reported millisecond process, belonging to the interconversion of both intermediates, can be observed in our single-molecule multiparameter fluorescence detection histograms.