The Persistent Effect of Initial Substrate Protein Conformation on Productive Folding by Groel-Groes

Abstract

The GroEL/GroES chaperonin system is required for the assisted folding of many essential proteins. In order to initiate a folding reaction, an open GroEL ring must capture a non-native substrate protein and encapsulate the protein inside an enclosed cavity that is formed when ATP and GroES bind to the same, substrate protein-occupied ring. Previous work demonstrated that the GroEL-dependent folding of highly recalcitrant proteins like RuBisCO is enhanced by two phases of structural disruption prior to the initiation of folding: a binding-driven unfolding phase upon substrate protein capture by a GroEL ring, and a forced expansion driven by the movement of the GroEL apical domains upon ATP binding. The overall impact, timing and significance of these unfolding events, particularly with regard to their coordination with GroES binding and substrate protein encapsulation, has remained controversial. Here we demonstrate that the conformation of a substrate protein at the moment of release into the GroEL-GroES cavity directly affects the efficiency of subsequent folding events. We first examined the conformation of non-native RuBisCO as it interacts with a GroEL variant possessing a substantially reduced ability to support folding. using a combination of resonance energy transfer and protease protection, we find that the non-native RuBisCO monomer adopts a less unfolded conformation upon initial capture by the GroEL variant. Subsequent binding of ATP results in a reduced forced expansion of RuBisCO by this GroEL variant. Despite efficient binding of GroES to a RuBisCO-occupied ring, however, the compaction of the folding intermediate during encapsulation is also substantially reduced. In combination, these deficits in initial structural alteration appear to result in a marked reduction in productive folding, suggesting a crucial role of these GroEL-dependent manipulations in preparation of the substrate protein for the folding process.