Abstract
Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all β-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2–3 kcal mol−1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both β-sheets, rather than by the location of the terminal strands in the ribosome tunnel.
Highlights
The relationship between the folding of domains in isolation and cotranslational folding on the ribosome has been investigated in recent years using a range of methods [1,2] including studies that have compared in vitro folding with folding in vivo [3,4,5,6,7,8,9,10,11,12]
Using MATH, an all β-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome
We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both β-sheets, rather than by the location of the terminal strands in the ribosome tunnel
Summary
The relationship between the folding of domains in isolation and cotranslational folding on the ribosome has been investigated in recent years using a range of methods [1,2] including studies that have compared in vitro folding with folding in vivo [3,4,5,6,7,8,9,10,11,12]. 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]
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