Transient misfolding dominates multidomain protein folding

Abstract

Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

“…Misfolding simulations of multidomain proteins such as titin suggest a role for domain swapping and/or self-similar amyloid-like interactions in misfolding and aggregation (75,76), and these studies have now been extended to HγD (66). In our predicted oligomerizing structure, strand 14 is sandwiched between two homologous β-strands, one belonging to the native structure (strand 13) and the other belonging to another domain or protein (strand 1).…”

A glass menagerie of low complexity sequences

“…lacking discrete stoichiometry) clusters, or phases, with critical concentrations for nucleation [1,13]. The analogous chain reaction for structured proteins, called “domain swapping”, renders tandem repeat multidomain proteins particularly susceptible to form solid phases -- that is, to aggregate [11,14]. The intrinsic polyvalency of LCSs also explains their well-documented promiscuity: disordered proteins have more binding partners than structured proteins; interact aberrantly with other proteins, leading to dosage-sensitive proteotoxicity [15]; and are enriched in the insoluble fractions of proteomes [16].…”

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

“…Proline residues play a prominent role in protein folding [1,2], protein mis-folding, and aggregation [3]. They are key to attaining the functional state of proteins [4].…”