(A) Our work on competing folding pathways, using time-resolved FRET measurements to study the folding of monellin, has revealed features of folding reactions that are likely to be generally relevant to how proteins fold. (1) There are multiple pathways and there are multiple steps on each pathway. Structural events occur in fast and slow phases on each pathway. (2) Structural events, such as core consolidation and β-sheet formation, can happen independently of each other. (3) A particular structural event may happen in molecules in which other structural events may or may not have already occurred. A structural event may occur in the fast phase on one pathway and in the slow phase on another pathway. (4) The rate constants of structure formation along different pathways involving different regions are the same. This suggests that the underlying physicochemical interactions that act cooperatively during the different stages of folding are similar, irrespective of the structure-forming part. It should, however, be noted that the nature of the barriers that dictate the relative fluxes of molecules on the parallel pathways is yet to be understood. (5) Very importantly, these results clearly contradict the notion that structure accumulation during folding must occur in a unique manner and on a single defined pathway. (Bhatia et al., 2021).

(B) Folding may switch from one pathway to an alternative pathway upon a change in folding conditions. Using hydrogen exchange-mass spectrometry measurements to study the folding of the heterodimeric protein, double chain monellin, the structural differences between the pathways of folding in two different conditions have been delineated. The major difference in the pathways is that β2 has not acquired any structure that is protective against hydrogen exchange in the intermediate formed on one pathway, but has acquired significantly protective structure in the intermediate formed on the alternative pathway. The intermediates on the two pathways also differ in the extent to which the a helix and the rest of the β-sheet have acquired protective structure. Hence, the sequence of structural events is different on the two alternative pathways (Bhattacharjee and Udgaonkar, 2022).

(C) Protein misfolding and aggregation appear to originate from partially unfolded conformations that are sampled through fluctuations of the native protein. It has been a challenge to characterize these fluctuations, under native like conditions. We have studied the conformational dynamics of the full-length (23-231) mouse prion protein under native conditions, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS). The slowest fluctuations could be associated with the folding of the unfolded state to an intermediate state, by the use of microsecond mixing experiments. The two faster fluctuations observed by PET-FCS, could be attributed to fluctuations within the native state ensemble. The addition of salt which is known to initiate aggregation of the protein, resulted in an enhancement in the time scale of fluctuations in the core of the protein, with  α2-α3 dynamics being modulated more than α1-α3 dynamics (Goluguri et al., 2019).

(D) It has been difficult to monitor structural changes in the monomeric units, as a protein misfolds and oligomerizes. Using intra-molecular FRET measurements, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, as the monomeric units transformed into large oligomers OL, which then dissociates into small oligomers OS. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL, and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in OL and OS. This experimental approach can be useful for testing the effect of pathogenic mutations and inhibitors on the local misfolding kinetics (Sengupta and Udgaonkar, 2019).

(E) Identifying the mechanisms of the initiation of conformational conversion during the aggregation of the prion protein has been a long-standing challenge. We have shown that two sets of evolutionarily conserved residues (a) Pro136, Pro157 and Pro164, and (b) Tyr168, Phe174 and Tyr217, act as gatekeepers that impede misfolding. When these residues are mutated to Ala, misfolding is accelerated up to nearly 1000-fold. Hydrogen exchange mass spectrometry measurements have identified several partially unfolded forms (PUFs) in equilibrium with the native state. In PUF2*, α1 and the spatially adjacent  N-terminal end of α2 have become disordered, while in  PUF2**, the β2-α2 loop and the C-terminal end of α3 have become disordered. The Pro to Ala mutations, and the Aromatic residue to Ala mutations lower the free energy of unfolding of N to PUF2* and PUF2**, respectively. Hence, misfolding can commence and proceed  in multiple ways from structurally distinct precursor conformations (Pal and Udgaonkar, 2022, 2023).