Figure 3.

GroES and ATP binding to a Rubisco-occupied trans ring results in forced conformational expansion, followed by compaction, of the non-native protein. (a) Schematic of a FRET experiment designed to examine conformational changes in a non-native substrate protein upon encapsulation beneath GroES on the trans ring of an ADP bullet. (b) GroES binding and substrate encapsulation results in an initial rapid expansion of the non-native Rubisco intermediate, followed by a compaction. Substrate-bound complexes in the presence of excess GroES were rapidly mixed in a stopped-flow apparatus (60 nM) with ATP (1 mM). For these measurements, the donor probe was fluorescein and the acceptor was rhodamine. Each FRET trace is the average of n = 10 replicates of matched experimental pairs, calculated from donor-only (Rubisco 454-F) and donor-acceptor (Rubisco 454-F/58-R) samples. The inset shows the FRET change over the first second of data. The rising phase in the observed FRET signal reflects GroES binding and substrate-protein encapsulation and compaction³⁵. (c) The observed rate constants for the initial rapid drop in FRET efficiency (k_obs) and the subsequent rise upon GroES binding (k_ES) for a series of experiments at different concentrations of GroES are shown. In each case, a fixed concentration of the Rubisco-bound GroEL ADP bullets (60 nM) and ATP (1 mM) was used, whereas the total concentration of GroES was varied.