Population of on-pathway intermediates in the folding of ubiquitin.
The role that intermediate states play in protein folding is the subject of intense investigation and in the case of ubiquitin has been controversial. We present fluorescence-detected kinetic data derived from single and double mixing stopped-flow experiments to show that the F45W mutant of ubiquitin (WT*), a well-studied single-domain protein and most recently regarded as a simple two-state system, folds via on-pathway intermediates. To account for the discrepancy we observe between equilibrium and kinetic stabilities and m-values, we show that the polypeptide chain undergoes rapid collapse to an intermediate whose presence we infer from a fast lag phase in interrupted refolding experiments. Double-jump kinetic experiments identify two direct folding phases that are not associated with slow isomerisation reactions in the unfolded state. These two phases are explained by kinetic partitioning which allows molecules to reach the native state from the collapsed state via two possible competing routes, which we further examine using two destabilised ubiquitin mutants. Interrupted refolding experiments allow us to observe the formation and decay of an intermediate along one of these pathways. A plausible model for the folding pathway of ubiquitin is presented that demonstrates that obligatory intermediates and/or chain collapse are important events in restricting the conformational search for the native state of ubiquitin.
Crespo
Maria D
MD
Centre for Biomolecular Sciences, School of Chemistry, University Park, Nottingham NG7 2RD, UK.
Simpson
Emma R
ER
Searle
Mark S
MS
D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics
D005656:Fungal Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D011392:Proline