Passage
Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is one of the most abundant enzymes on Earth. In carbon-fixing organisms, it consumes CO₂ to carboxylate the sugar ribulose-1,5-bisphosphate (Ru1,5BP) , and forms two molecules of 3-phosphoglycerate (3PG) as a product. The functional form of rubisco from the bacterium Rhodospirillum rubrum is a noncovalent homodimer composed of two 51 kDa monomers. Each subunit requires a magnesium ion as a prosthetic group in the active site.Rubisco is often used as a model for protein folding studies. On their own, rubisco monomers typically cannot fold to completion and instead are trapped in a form known as a "kinetic intermediate." These kinetically trapped monomers cannot dimerize correctly and are prone to aggregation.Scientists interested in studying protein folding dynamics labeled the rubisco kinetic intermediate with a set of fluorophores known as a FRET (fluorescence resonance energy transfer) pair, with one fluorophore at the N-terminus and the other at the C-terminus. A FRET signal occurs when the emission spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of the other (the acceptor) . When the two fluorophores are sufficiently close to each other, the donor can transfer energy from the light it absorbs directly to the acceptor, causing the acceptor to emit light when the donor absorbs light.The GroEL/ES complex is a chaperone protein that binds and releases misfolded proteins, hydrolyzing ATP each time the protein is released. A misfolded protein may undergo multiple rounds of binding and release before adopting its correct conformation, or it may never fold correctly and instead be targeted for destruction (Figure 1) .
Figure 1 Representation of GroEL/ES-mediated protein foldingResearchers diluted the labeled kinetic intermediate into a refolding buffer in the presence or absence of 200 nM GroEL/ES and then monitored the FRET signal over time. The results are shown in Figure 2.
Figure 2 FRET signal of labeled rubisco with and without GroEL
Lin Z, Rye HS. Expansion and compression of a protein folding intermediate by GroEL. Mol Cell. 2004;16(1) :23-34.
-Which of the following experiments, performed in a closed system, could confirm that GroEL/ES successfully facilitated proper rubisco folding?

Question

Passage
Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is one of the most abundant enzymes on Earth. In carbon-fixing organisms, it consumes CO₂ to carboxylate the sugar ribulose-1,5-bisphosphate (Ru1,5BP) , and forms two molecules of 3-phosphoglycerate (3PG) as a product. The functional form of rubisco from the bacterium Rhodospirillum rubrum is a noncovalent homodimer composed of two 51 kDa monomers. Each subunit requires a magnesium ion as a prosthetic group in the active site.Rubisco is often used as a model for protein folding studies. On their own, rubisco monomers typically cannot fold to completion and instead are trapped in a form known as a "kinetic intermediate." These kinetically trapped monomers cannot dimerize correctly and are prone to aggregation.Scientists interested in studying protein folding dynamics labeled the rubisco kinetic intermediate with a set of fluorophores known as a FRET (fluorescence resonance energy transfer) pair, with one fluorophore at the N-terminus and the other at the C-terminus. A FRET signal occurs when the emission spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of the other (the acceptor) . When the two fluorophores are sufficiently close to each other, the donor can transfer energy from the light it absorbs directly to the acceptor, causing the acceptor to emit light when the donor absorbs light.The GroEL/ES complex is a chaperone protein that binds and releases misfolded proteins, hydrolyzing ATP each time the protein is released. A misfolded protein may undergo multiple rounds of binding and release before adopting its correct conformation, or it may never fold correctly and instead be targeted for destruction (Figure 1) .

Figure 1 Representation of GroEL/ES-mediated protein foldingResearchers diluted the labeled kinetic intermediate into a refolding buffer in the presence or absence of 200 nM GroEL/ES and then monitored the FRET signal over time. The results are shown in Figure 2.

Figure 2 FRET signal of labeled rubisco with and without GroEL
Lin Z, Rye HS. Expansion and compression of a protein folding intermediate by GroEL. Mol Cell. 2004;16(1) :23-34.
-Which of the following experiments, performed in a closed system, could confirm that GroEL/ES successfully facilitated proper rubisco folding?

Quizplus · Accepted Answer

11ecba2d_0e23_94a8_a3bf_1dbf22a6680f_MD0008_00 The function of a protein is determined by its three-dimensional folded form. A properly folded protein will be active, so an assay of its activity can reveal whether correct folding has been achieved. GroEL/ES is a chaperone protein that, by definition, helps other proteins to fold. Figure 2 shows that rubisco undergoes a conformational change in the presence of GroEL/ES, but it is possible that the form facilitated by GroEL/ES is still not the correctly folded, functional form. To confirm that the functional form has been produced, researchers could assay rubisco activity. The passage states that rubisco carboxylates Ru1,5BP and hydrolyzes it to produce two 3PG molecules as products. Carboxylation is the attachment of CO₂ to another molecule. In an open system, CO₂ from the atmosphere can dissolve into solution to replace CO₂ consumed by the reaction. However, the question specifies that the experiment is carried out in a closed system, in which matter cannot be exchanged with the environment. In a closed system containing correctly folded rubisco and Ru1,5BP, the concentration of dissolved CO₂ will decrease over time as it is consumed by the reaction. Therefore, CO₂ consumption under these conditions indicates that rubisco is properly folded. (Choice B) The ability of rubisco to dimerize is an indication that it is folded correctly. However, SDS gels denature proteins and destroy quaternary structure, such as noncovalent dimerization, so they cannot be used to assess proper folding. (Choice C) GroEL/ES hydrolyzes ATP to ADP and inorganic phosphate (P_i) each time it releases a bound protein, so the P_i concentration increases over time. In addition, the passage states that a misfolded protein may require multiple rounds of GroEL/ES binding, and that it may never fold correctly. A change in P_i concentration does not guarantee correct protein folding. (Choice D) The K_d of magnesium binding to rubisco would help confirm correct folding, but magnesium binding to GroEL/ES is not related to the conformation of rubisco. Educational objective: The function of a protein is tied directly to its native, three-dimensional form. Activity assays can confirm that a protein has folded correctly.

Passage Ribulose-1,5-Bisphosphate Carboxylase/oxygenase (Rubisco) Is One of the Most Abundant Enzymes