Effect of circular permutations on transient partial unfolding in proteins

Abstract

Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the threedimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins. We constructed three circular permutants, CP18, CP37, and CP87, with the new N-termini at residue 18, 37, and 87, respectively, and probed transient partial unfolding by native-state proteolysis. The new termini in CP18, CP37, and CP87 are within, near, and distal to the Met20 loop, which is known to be dynamic and also part of the region that undergoes transient unfolding in wild-type DHFR. The stabilities of both native and partially unfolded forms of CP18 are similar to those of wild-type DHFR, suggesting that the influence of introducing new termini in a dynamic region to the protein is minimal. CP37 has a significantly more accessible partially unfolded form than wild-type DHFR, demonstrating that introducing new termini near a dynamic region may promote transient partial unfolding. CP87 has significantly destabilized native and partially unfolded forms, confirming that modification of the folded region in a partially unfolded form destabilizes the partially unfolded form similar to the native form. Our findings provide valuable guidelines to control transient partial unfolding in designing circular permutants in proteins.

Metal‐binding and circular permutation‐dependent thermodynamic and kinetic stability of azurin

“…CP of proteins allows changing the order of secondary structural elements, keeping the 3D structure of the protein intact, and introducing new N-and C-termini. CP has been performed to see its effects on stability, 3,14,20,78 folding-unfolding pathways, 14,[16][17][18][19]21,22,[26][27][28] and biological activity 15,[78][79][80][81][82][83][84] 6) are represented as the free energy diagrams of WT and cpN42, displaying the activation barriers and thermodynamic energy differences (Figure 7). We find that the CP of azurin is thermodynamically less stable than the WT-azurin protein ($15 kJ/mol) both in apo-and holoforms.…”

“…3,14,16,17,[19][20][21] There are several experimental and computational studies where CP has led to changes in the protein folding and unfolding pathways and/or the transition state structures involved. 14,[16][17][18][19][20][21][22][23][24][25][26][27][28] An exhaustive thermodynamic, kinetic, and ϕ-value analysis of CPs of S6 protein shows competing nuclei participating in folding, depending on the exact protein sequence. 16 Despite these crucial investigations on comparing the stability and folding pathways of proteins with their CPs, similar studies are yet to be undertaken on proteins binding metals or small molecule cofactors.…”

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome

“…Circular permutants (CPs) of isolated domains have been extensively studied to investigate the relationship between protein topology, chain connectivity, and folding pathways [30], [31], [32], [33], [34], [35]: the permuted protein is covalently linked at the N and C termini, and new termini are generated elsewhere in the sequence, usually in a loop region [36]. This allows retention of the same amino acid composition and chain length as wild-type, but alters the connectivity of secondary structure elements; this in turn may lead to alterations in protein stability [37], [38], [39], enzyme activity [40], [41], and folding pathway [42], [43], [44].…”