Unraveling Folding Pathways and Kinetics Transition of T4 Lysozyme with High Temporal Resolution by Single Molecule FRET

Abstract

Protein folding is directly connected to protein function, and miss folding events could lead to disease. Currently, one of the challenges of understanding protein folding is to determine the number of meta stable or excited intermediate states and their corresponding kinetic pathways during folding transitions given the large number of possible accessible conformers. Therefore, we combine the power of single molecule FRET, with site specific labeling, filtered fluorescence correlation spectroscopy (fFCS), multiparameter fluorescence detection (MFD) and ensemble time correlated single photon counting to determine the folding and unfolding kinetics and the corresponding reaction pathways of T4 Lysozyme (T4L) under various chemical denaturation conditions (Urea, GdmCl, and pH) with sub microsecond resolution.MFD, a single molecule technique, allows the direct observation of populations; even when low populated (<10%). fFCS, a recently developed modification of the standard FCS, uses the fluorescence lifetime and anisotropy decays to filter the signal and properly weight the contribution of each photon to the corresponding population in an heterogeneous solution. The weighted time dependent signal with specific spectral window and polarization is correlated to obtain all possible auto-correlation and cross-correlation curves. Of great utility is the cross-correlation curve because it resolves, with maximum contrast, the anticorrelated behavior of the interconversion between populations.Using the afore mentioned toolbox, we observe that each denaturant results in different folding and unfolding pathways in T4L. Furthermore, the addition of surfactant (Tween 20) changes dramatically the reaction coordinate in the folding and unfolding energy landscape even in the same denaturant conditions. One of the major differences observed is that pH denaturation showed the accumulation of an intermediate state where most likely the N terminal domain is partially unfolded while the C terminal domain remains folded.