Protein misfolding: optional barriers, misfolded intermediates, and pathway heterogeneity.
To investigate the character and role of misfolded intermediates in protein folding, a recombinant cytochrome c without the normally blocking histidine to heme misligation was studied. Folding remains heterogeneous as in the wild-type protein. Half of the population folds relatively rapidly to the native state in a two-state manner. The other half collapses (fluorescence quenching) and forms a full complement of helix (CD) with the same rate and denaturant dependence as the fast folding fraction but then is blocked and reaches the native structure (695nm absorbance) much more slowly. The factors that transiently block folding are not intrinsic to the folding process but depend on ambient conditions, including protein aggregation (f(concentration)), N terminus to heme misligation (f(pH)), and proline mis-isomerization (f(U state equilibration time)). The misfolded intermediate populated by the slowly folding fraction was characterized by hydrogen exchange pulse labeling. It is very advanced with all of the native-like elements fairly stably formed but not the final Met80-S to heme iron ligation, similar to a previously studied molten globule form induced by low pH. To complete final native state acquisition, some small back unfolding is required (error repair) but the misfolded intermediate does not revisit the U state before proceeding to N. These properties show that the intermediate is a normal on-pathway form that contains, in addition, adventitious misfolding errors that transiently block its forward progress. Related observations for other proteins (partially misfolded intermediates, pathway heterogeneity) might be similarly explained in terms of the optional insertion of error-dependent barriers into a classical folding pathway.
Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA. mmg@hx2.med.upenn.edu <mmg@hx2.med.upenn.edu>
Mallela M G
MM
Krishna
Yan
Y
Lin
S Walter
SW
Englander
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013997:Time Factors
D045304:Cytochromes c