The Response of Greek Key Proteins to Changes in Connectivity Depends on the Nature of Their Secondary Structure

Abstract

What governs the balance between connectivity and topology in regulating the mechanism of protein folding? We use circular permutation to vary the order of the helices in the all-α Greek key protein FADD (Fas-associated death domain) to investigate this question. Unlike all-β Greek key proteins, where changes in the order of secondary structure cause a shift in the folding nucleus, the position of the nucleus in FADD is unchanged, even when permutation reduces the complexity significantly. We suggest that this is because local helical contacts are so dominant that permutation has little effect on the entropic cost of forming the folding nucleus whereas, in all-β Greek key proteins, all interactions in the nucleus are long range. Thus, the type of secondary structure modulates the sensitivity of proteins to changes in connectivity.

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

“…There have been a number of studies of Greek-key proteins that have given support to an obligate-critical nucleus model such as that we propose here: The Ig domain titin I27 (I27) and the evolutionarily unrelated fnIII domains, TNfn3, FNfn10, and CAfn2 share a structurally equivalent obligate nucleus, comprising key residues located in the four central strands, which establishes the topology of these complex Greek-key domains [60], [61], [62], [63], [64]. Similar results were seen for the all-beta Greek key protein S6, and for an all-alpha Greek key death domain [35], [43].…”

“…The vectorial emergence of the peptide chain from the ribosome tunnel prompts the question of how circular permutation of a protein affects cotranslational folding. 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].…”

Slowest-first protein translation scheme: Structural asymmetry and co-translational folding

“…To experimentally test the slowest-first mechanism, we suggest studying CTF of multiple proteins with , which differ in and . In particular, we propose to use proteins whose sequences are related by “circular permutation,” while having identical structures ( 85 , 102 , 103 , 104 ). Circular permutants with opposite structural asymmetry, as the example in Fig.…”

“…Consequentially, on average, b strands take longer to form contacts, and suffer a greater entropic penalty to stability (115). There is ample evidence linking topology to folding rate; structures with more long-range contacts tend to fold slower (73,104,(116)(117)(118)(119)(120)(121). Experimental support for this topological effect is found in designed peptides that fold close FIGURE 5 Secondary structure for nuclear transport factor 2 H66A mutant (PDB: 1ASK ( 106)) and a circular permutant, 1ASK-CP67, which may fold faster during translation.…”

Structural asymmetry along protein sequences and co-translational folding

“…To experimentally test the slowest-first mechanism, we suggest studying CTF of multiple proteins with R ≈ 1, which differ in asym α and asym β . In particular, we propose to use proteins whose sequences are related by circular permutation, while having identical structures [65][66][67][68]. Circular permutants with opposite structural asymmetry, as the example in Fig.…”