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 conclusions can be drawn based on the data from Figure 2?

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 conclusions can be drawn based on the data from Figure 2?

Quizplus · Accepted Answer

11ecba2d_0e20_8765_a3bf_91e45857305c_MD0008_00 Proteins fold into three-dimensional structures. In the folded form, amino acids that are far apart in the primary structure may be close together in space. The proximity of amino acids to each other in a folded protein can be probed by techniques such as FRET. The passage states that a FRET signal occurs when two fluorophores with overlapping excitation and emission spectra (FRET pair) are near each other. It also states that the N- and C-termini of rubisco were labeled with a FRET pair. Figure 2 shows that in the absence of GroEL/ES, a relatively strong FRET signal is observed and remains over the course of the experiment, suggesting that the rubisco kinetic intermediate must be folded such that the termini are near each other. In the presence of GroEL/ES, the FRET signal is present initially, which is consistent with the fact that initially rubisco was in the kinetic intermediate form. However, over the course of the experiment the signal decreases. This indicates that the fluorophores are being separated from each other, and therefore GroEL/ES causes the termini of the kinetic intermediate to separate. (Choice A) The FRET donor absorbs light and transfers the energy to the acceptor. GroEL does not transfer energy to the FRET donor. (Choice C) The GroEL/ES complex is not labeled with either part of the FRET pair. Therefore, it does not have overlapping excitation or emission spectra with rubisco. (Choice D) GroEL/ES increases the distance between rubisco termini, causing a decrease in the FRET signal. Educational objective: Proteins fold into three-dimensional structures. Amino acids that are far away from each other in the linear sequence (primary structure) may be brought close together in space when the protein folds.

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