Skip to main content
. Author manuscript; available in PMC: 2013 Aug 15.
Published in final edited form as: Nat Struct Mol Biol. 2008 Mar 2;15(3):303–311. doi: 10.1038/nsmb.1394

Figure 2.

Figure 2

Non-native Rubisco bound to the trans ring of an ADP bullet is less susceptible to protease digestion and shows reduced chemical modification of internal cysteine residues. (a) Fluorescently labeled Rubisco (58-F; 100 nM) was denatured and bound to either ADP bullets or apoGroEL (labeled trans and apo, respectively; 120 nM in each case), treated with chymotrypsin for the indicated times and then analyzed by SDS-PAGE and laser-excited, fluorescence gel scanning. Partial digestion fragments that appear uniquely well represented in either the apo or trans digestion experiments are indicated by a square bracket and asterisks. (b) The amount of full-length, fluorescent Rubisco was quantified for three independent protease experiments, and the average of these replicates is plotted as a function of digestion time (error bars show one s.d.). The t1/2 for the disappearance of full-length Rubisco bound to apoGroEL and an ADP-bullet trans ring are 0.6 min and 1.3 min, respectively. (c) The internal structure of Rubisco monomers bound to apoGroEL and the ADP-bullet trans ring was probed with a small, highly reactive coumarin maleimide dye (CPM) that selectively modifies exposed cysteine residues60. Only a single surface-exposed cysteine residue at position 58 (out of a total of five cysteine residues in wild-type Rubisco) is modified in native Rubisco (labeled Native (58C); 500 nM). The graph shows the total level of CPM fluorescence detected in the Rubisco sample following analysis by reverse-phase HPLC. When non-native Rubisco (500 nM) is bound to either apoGroEL (apo; 600 nM) or an ADP-bullet trans ring (trans; 600 nM), the internal Rubisco cysteine residues become highly reactive toward CPM. However, the reactivity of internal cysteine residues in Rubisco bound to apoGroEL is significantly greater than the trans ring–bound sample (P < 0.003 based on a paired t-test with n = 3 replicates; error bars show one s.d.). The maximum level of internal CPM incorporation expected under the solution conditions used is also shown (indicated by a dashed line, labeled five-fold 58C) and was estimated by assuming that the internal Rubisco cysteine residues in unfolded Rubisco would be as exposed and reactive as the surface-exposed 58C site.