Abstract
Abstract To date, about 700 knotted proteins have been identified in the protein data bank (PDB). How and why these protein adopt knotted backbone topologies remain elusive. HP0242 is a hypothetical protein from Helicolbacter pylori which is homodimeric and contains a novel interwined topology (a pseudo trefoil 31 knot); it will be properly knotted when two monomers are concatenated. HP0242 is an ideal model system for studying knotted protein folding. While far-UV circular dichroism (CD) spectroscopy with both in-house and synchrotron radiation light sources identifies the existence of a highly populated folding intermediate during chemical denaturation, intrinsic fluorescence spectroscopy only probes the native (N) to intermediate (I) state transition because HP0242 only has one tryptophan residue and it is likely that the local structure around the tryptophan side chain becomes fully solvent exposed while a significant part of the structure remains folded in the intermediate state. We therefore design two Phe-to-Trp mutants to monitor folding events at the other sites of HP0242. Together with small angle X-ray scattering (SAXS) and dynamic light scattering (DLS), which report on the changes of radius of gyration (Rg) and radius of hydration (Rh), respectively, as a function of denaturant concentration, we have established the sequential folding processes of HP0242 during chemical denaturation. 6 In light the of the emerging interests in employing theoretical modeling approaches to delineate the folding pathways of knotted proteins, our results provide key experimental evidence of sequential folding events of HP0242 which can be validated in the theoretical models.