Figure 1.

Non-native Rubisco is more compact when bound to the trans ring of an ADP bullet than when bound to apoGroEL. (a) The conformation of a substrate protein bound to either apoGroEL or the trans ring of an ADP bullet can be investigated with intramolecular FRET, where the distance between two exogenous fluorescent probes attached to different parts of the substrate protein (indicated schematically as colored circles labeled ‘A’ and ‘D’) is examined. (b) Steady-state FRET measurement of the distance between the N- and C-terminal domains of a non-native Rubisco monomer (100 nM) bound to an ADP-bullet trans ring and apoGroEL (120 nM in each case). The C-terminal domain of a previously described Rubisco variant³⁵ was labeled with a donor fluorophore and the N-terminal domain was labeled with an acceptor fluorophore. For these measurements, the donor probe was 5-(2-acetamidoethyl) aminonaphthalene-1-sulfonate (AEDANS; 454-ED) and the acceptor was fluorescein (58-F). The donor-emission spectra of donor-only (labeled apo-D and trans-D, respectively) and donor-acceptor (labeled apo and trans, respectively) samples are shown. (c) Time-resolved donor-intensity decays of the samples described in b. The donor-emission decay of donor-only and donor-acceptor samples, labeled as above, and the instrument response function (IRF) are shown. The average intraprobe distance was quantified from both steady-state and time-resolved FRET measurements (b, inset).