Abstract
DehI is a homodimeric haloacid dehalogenase from Pseudomonas putida that contains the most complex 61 Stevedore’s protein knot within its folding topology. To examine how DehI attains such an intricate knotted topology we combined far-UV circular dichroism (CD), intrinsic fluorescence spectroscopy and small angle X-ray scattering (SAXS) to investigate its folding mechanism. Equilibrium unfolding of DehI by chemical denaturation indicated the presence of two highly populated folding intermediates, I and I’. While the two intermediates vary in secondary structure contents and tertiary packing according to CD and intrinsic fluorescence, respectively, their overall dimension and compactness are similar according to SAXS. Three single-tryptophan variants (W34, W53, and W196) were generated to probe non-cooperative unfolding events localized around the three fluorophores. Kinetic fluorescence measurements indicated that the transition from the intermediate I’ to the unfolded state is rate limiting. Our multiparametric folding analyses suggest that DehI unfolds through a linear folding pathway with two distinct folding intermediates by initial hydrophobic collapse followed by nucleation condensation, and that knotting precedes the formation of secondary structures.
Highlights
Knotted proteins have in recent years attracted tremendous attentions from biophysicists because of their abilities to spontaneously fold into natively knotted conformations despite the apparent structural complexities[1,2,3]
Similar to YibK, these Gordian knotted proteins exhibit parallel folding pathways with distinct folding intermediates that are hyperfluorescent in the case of UCH-L331, and are highly populated during equilibrium unfolding in the case of UCH-L132
The far-UV circular dichroism (CD) spectrum indicates that DehI is highly helical with two well-defined peak signals at 208 and 222 nm with a predicted α-helical content of 54.9%, which is consistent with the crystal structure of DehI that is 59.5% α-helical (Figure S1B)
Summary
Knotted proteins have in recent years attracted tremendous attentions from biophysicists because of their abilities to spontaneously fold into natively knotted conformations despite the apparent structural complexities[1,2,3]. Similar to YibK, these Gordian knotted proteins exhibit parallel folding pathways with distinct folding intermediates that are hyperfluorescent in the case of UCH-L331, and are highly populated during equilibrium unfolding in the case of UCH-L132. A linker (residues 131–165) forming a number of hydrogen bonds and salt-bridges in between the two domains connects them to complete the 61 knotted topology (Fig. 1) This is reminiscent to the construction of an engineered protein knot by linking two unknotted subunits within a homodimeric HP0242 protein by means of gene duplication[29,47]. Through comparison of the spectroscopic characteristics, thermodynamic and kinetic parameters of single-tryptophan variants with those of the wild type (wt), a linear folding pathway with two distinct folding intermediates was proposed for DehI
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