Chaperonins Resculpt Folding Free Energy Landscapes To Avoid Kinetic Traps and Accelerate Protein Folding Xin Zhang, Jeffery W. Kelly PII: DOI: Reference:
S0022-2836(14)00280-0 doi: 10.1016/j.jmb.2014.06.001 YJMBI 64474
To appear in:
Journal of Molecular Biology
Please cite this article as: Zhang, X. & Kelly, J.W., Chaperonins Resculpt Folding Free Energy Landscapes To Avoid Kinetic Traps and Accelerate Protein Folding, Journal of Molecular Biology (2014), doi: 10.1016/j.jmb.2014.06.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Chaperonins Resculpt Folding Free Energy Landscapes To Avoid Kinetic Traps and Accelerate Protein Folding
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Xin Zhang1,2 and Jeffery W. Kelly1,2,3*
Departments of Chemistry, 2Molecular and Experimental Medicine, and 3the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA
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To whom correspondence should be addressed: [email protected] 858-784-9880
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The manuscript entitled “Active cage mechanism of chaperone-assisted protein folding demonstrated at single molecule level” by Gupta et al. in this issue of Journal of Molecular Biology provides compelling evidence that chaperonins actively fold proteins by altering their folding energetics1. For simple proteins exhibiting relatively smooth folding free energy funnels that lack deep kinetic traps (Figure 1A), the GroEL/ES chaperonins do not appear to alter the rate of folding measurably, probably because there are no kinetically trapped intermediates to bind to and untrap through conformational conversion or associated transition states to lower. In contrast, for complex proteins, exhibiting rugged free energy folding funnels and deep kinetic traps, chaperonins can alter the activation free energy of folding by ≈ an order of magnitude or by ≥ 1 kcal/Mol or more (Figure 1B). In other words, chaperonins can catalyze protein folding in addition to minimizing reversible and irreversible protein aggregation, by altering the free energy folding funnel by virtue of the physical chemical microenvironment within the chaperonin. The resulting rate acceleration allows folding to occur at a biologically relevant time scale.
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There are three competing hypotheses about how chaperonins function in prokaryotes and eukaryotic cells. The “passive cage” or “Anfinsen cage” hypothesis posits that chaperonins merely sequester individual proteins in a water-filled cylinder at effectively infinite dilution, thus the client protein folds at the same rate as in buffer and aggregation is prevented. In contrast, the “active cage” hypothesis suggests that beyond just preventing aggregation, the physical chemical attributes of the interior of these chaperonins can result in accelerated folding for a subset of client proteins. In the third “iterative annealing” model, the hypothesis is that chaperonins unfold misfolded proteins through iterative cycles of binding and release, with folding occurring either inside or outside of the cylinder. In this model, accelerated folding results from active unfolding of kinetically trapped conformations that partition between subsequent folding and misfolding processes. The transient encapsulation of the protein client within the cylinder is considered to be a by-product of this mechanism. There are several experimental challenges associated with differentiating these potential mechanisms of chaperonin action, realizing that the mechanism that applies could be protein-client specific. First, it is ideal to establish conditions that eliminate reversible and irreversible aggregation during quantitative spontaneous folding in buffer to differentiate these mechanisms. Second, if folding could be demonstrated to occur by a single encapsulation event, this is ideal for eliminating the “iterative annealing” mechanism for a particular client protein. To establish conditions of spontaneous, quantitative refolding, where reversible and irreversible aggregation is not occurring, the authors resorted to single molecule intermolecular fluorescence resonance energy transfer (FRET) measurements at low protein client concentrations. The client or substrate protein employed by the authors is a double mutant of the 41 kDa maltose binding protein (DM-MBP), a monomeric protein that folds slowly (t1/2=35 min at 25 °C) but in quantitative yield. The authors showed that GuHCl-denatured DM-MBP labeled with a small molecule FRET donor at position 312 that was mixed with an equal concentration of GuHCl-denatured DM-MBP labeled at
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position 312 with a small molecule FRET acceptor (50 pM each) did not exhibit a crosscorrelation signal during the spontaneous folding reaction monitored by dual color fluorescence cross-correlation single molecule experiments. Spiking in 5 pM of DMMBP double labeled (DL) with a donor fluorophore at position 30 and an acceptor fluorophore at position 312, to simulate a non-covalent DM-MBP dimer, revealed an easily detected cross-correlation signal, indicating that less than 5% of an aggregate could be readily detected. Moreover, the authors also used fluorescence correlation spectroscopy to show that the number of DM-MBP acceptor labeled fluorophores (at position 312) remained constant over the folding period. This signal would be predicted to change with time if reversible or irreversible aggregation had occurred, providing additional strong evidence for the lack of aggregation during spontaneous folding.
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To facilitate folding in buffer vs. chaperonin-mediated folding rate comparisons, the authors utilized a single pair FRET approach (spFRET)–a single molecule approach. The DM-MBP(DL) FRET chaperonin client introduced above was used for these experiments. The spontaneous folding reaction was stopped at time points of interest by adding GroEL in the absence of ATP, which binds unfolded and misfolded DMMBP(DL), arresting additional folding by serving as a “holdase chaperone”. The natively folded ensemble of conformations exhibit a FRET efficiency of 0.72, whereas 40% of unfolded DM-MBP(DL) exhibits a FRET efficiency of 0.06, consistent with an expanded conformational ensemble. Quantification of the 0.06 and 0.72 FRET efficiencies enabled the authors to extract folding rates at a concentration of 100 pM DM-MBP(DL). The same approach was used to measure chaperonin-assisted DM-MBP(DL) folding rates, except that apyrase was added to deplete the folding reaction of ATP and to convert the chaperonin to a “holdase chaperone”, preventing additional folding during the measurement period. Notably, chaperonin-assisted folding was 5.6 fold faster than spontaneous DM-MBP(DL) folding. Single molecule fluorescence correlation spectroscopy revealed an analogous acceleration in chaperonin assisted folding. Ensemble FRET measurements at the same concentration revealed a 7.7 fold increase in the rate of chaperonin-assisted folding. When position 312 of DM-MBP was labeled only with the fluorescence FRET acceptor Atto655, this electronically excited fluorophore can transfer an electron to tryptophan side chains upon contact, which occurs in the unfolded ensemble. Photoinduced electron transfer coupled with fluorescence correlation spectroscopy was used by the authors to quantify the entropy in the dynamic folding intermediate of DM-MBP in the spontaneous folding reaction and in chaperonin-assisted folding. While binding of DM-MBP to GroEL restricts chain dynamics modestly, GroEL/ES encapsulation results in a marked restriction of chain flexibility, as reflected by the relaxation time of the DM-MBP folding intermediate going from 40 to 99 s. Thus the chemical environment of the chaperonin reduces chain entropy, which is one strategy to reduce the activation energy to get to the native state. That a reduction in chain entropy is key is supported by a previous publication by the Hartl/Hayer-Hartl lab demonstrating that strategic placement of disulfide bonds in MBP renders it a fast folder2.
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To scrutinize the “iterative annealing” model of chaperonin function, several experiments were performed, including employing a single ring GroEL/ES chaperonin variant to demonstrate that a single round of encapsulation inside the chaperonin cylinder is sufficient to achieve hastened DM-MBP folding in quantitative yield. Moreover, dramatically increasing the concentration of native GroEL/ES in the single molecule experiments relative to ensemble experiments did not slow folding kinetics of DM-MBP or decrease yield, which is best rationalized and likely can only be rationalized by encapsulation as the basis of folding catalysis. Under the conditions of the single molecule experiments, the authors present a logical argument that 80% of unfolded or misfolded DM-MBP is always encapsulated, while the remainder is bound to the apical domain of GroEL. Taken together, this is compelling evidence that folding occurs almost exclusively within GroEL/ES, if not entirely there.
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Additional striking evidence that folding and folding catalysis occurs by chaperoninmediated encapsulation of client proteins arises from converting GroEL, which has 42 negative charges on the wall of the cis cavity, to a chaperonin with a net charge of 0. This was accomplished by converting Glu252, Asp253, Glu255, Asp359, Asp361 and Glu363 to Lys residues through mutagenesis. Notably, this KKKKKK-GroEL ceases to be a folding catalyst for DM-MBP, and the photoinduced electron transfer coupled with fluorescence correlation spectroscopy no longer demonstrates that the mobility of the DM-MBP folding intermediate is reduced upon encapsulation. Importantly, the removal of the net negative charge converts the active GroEL/ES chaperonin to a passive chaperonin for the DM-MBP client protein.
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Both the Hartl/Hayer-Hartl lab3 and independently later the Rye lab4 published that RuBisCO refolding is also accelerated by GroEL/ES encapsulation, using a range of single molecule and ensemble measurements. Together they used single molecule experiments and related experiments to demonstrate beyond any reasonable doubt that reversible aggregation of RuBisCO does not occur under the conditions used to demonstrate chaperonin-mediated catalysis of RuBisCO folding. The Hartl/Hayer-Hartl lab also demonstrated recently that GroEL/ES also accelerates the folding of the tetrameric TIM-barrel protein DapA, an enzyme that catalyzes the formation of dihydropicolinic acid. GroEL/ES accelerates folding of DapA 30 fold and renders formation of the folded ensemble more segmental and less cooperative inside the cage relative to spontaneous DapA folding, as discerned by HD exchange experiments5. This mechanism lowers the high entropic component of the folding energy barrier associated with spontaneous folding. In summary, there is now compelling evidence from the manuscripts outlined here that chaperonins can act as an “active cage” for proteins that get stuck in a kinetic trap(s) during spontaneous folding. Interestingly, GroEL/ES functions as an active cage for DMMBP and as a passive cage for wild type MBP assisted folding. All of the data reported to date are consistent with a model for chaperonin-mediated folding catalysis wherein the chemical environment of the chaperonin reduces chain entropy destabilizing kinetically trapped folding intermediates, thus reducing the activation energy required to get to the
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Gupta, A. J., Haldar, S., Milicic, G., Hartl, F. U. & Hayer-Hartl, M. (2014). Active cage mechanism of chaperonin-assisted protein folding demonstrated at single molecule level. Journal of Molecular Biology 426, DOI: 10.1016/j.jmb.2014.04.018. Chakraborty, K., Chatila, M., Sinha, J., Shi, Q., Poschner, B. C., Sikor, M., Jiang, G., Lamb, D. C., Hartl, F. U. & Hayer-Hartl, M. (2010). Chaperonin-catalyzed rescue of kinetically trapped states in protein folding. Cell 142, 112-22. Brinker, A., Pfeifer, G., Kerner, M. J., Naylor, D. J., Hartl, F. U. & Hayer-Hartl, M. (2001). Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223-33. Lin, Z. & Rye, H. S. (2004). Expansion and compression of a protein folding intermediate by GroEL. Mol Cell 16, 23-34. Georgescauld, F., Popova, K., Gupta, A. J., Bracher, A., Engen, J. R., HayerHartl, M. & Hartl, F. U. (2014). GroEL/ES Chaperonin Modulates the Mechanism and Accelerates the Rate of TIM-Barrel Domain Folding. Cell 157, 922-34.
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native state, while preventing aggregation by folding within the cage or cylinder (Figure 1B)1; 2; 3; 4; 5.
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Figure 1
S0022-2836(14)00280-0 doi: 10.1016/j.jmb.2014.06.001 YJMBI 64474
To appear in:
Journal of Molecular Biology
Please cite this article as: Zhang, X. & Kelly, J.W., Chaperonins Resculpt Folding Free Energy Landscapes To Avoid Kinetic Traps and Accelerate Protein Folding, Journal of Molecular Biology (2014), doi: 10.1016/j.jmb.2014.06.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
PT
Chaperonins Resculpt Folding Free Energy Landscapes To Avoid Kinetic Traps and Accelerate Protein Folding
RI
Xin Zhang1,2 and Jeffery W. Kelly1,2,3*
Departments of Chemistry, 2Molecular and Experimental Medicine, and 3the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA
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To whom correspondence should be addressed: [email protected] 858-784-9880
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The manuscript entitled “Active cage mechanism of chaperone-assisted protein folding demonstrated at single molecule level” by Gupta et al. in this issue of Journal of Molecular Biology provides compelling evidence that chaperonins actively fold proteins by altering their folding energetics1. For simple proteins exhibiting relatively smooth folding free energy funnels that lack deep kinetic traps (Figure 1A), the GroEL/ES chaperonins do not appear to alter the rate of folding measurably, probably because there are no kinetically trapped intermediates to bind to and untrap through conformational conversion or associated transition states to lower. In contrast, for complex proteins, exhibiting rugged free energy folding funnels and deep kinetic traps, chaperonins can alter the activation free energy of folding by ≈ an order of magnitude or by ≥ 1 kcal/Mol or more (Figure 1B). In other words, chaperonins can catalyze protein folding in addition to minimizing reversible and irreversible protein aggregation, by altering the free energy folding funnel by virtue of the physical chemical microenvironment within the chaperonin. The resulting rate acceleration allows folding to occur at a biologically relevant time scale.
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There are three competing hypotheses about how chaperonins function in prokaryotes and eukaryotic cells. The “passive cage” or “Anfinsen cage” hypothesis posits that chaperonins merely sequester individual proteins in a water-filled cylinder at effectively infinite dilution, thus the client protein folds at the same rate as in buffer and aggregation is prevented. In contrast, the “active cage” hypothesis suggests that beyond just preventing aggregation, the physical chemical attributes of the interior of these chaperonins can result in accelerated folding for a subset of client proteins. In the third “iterative annealing” model, the hypothesis is that chaperonins unfold misfolded proteins through iterative cycles of binding and release, with folding occurring either inside or outside of the cylinder. In this model, accelerated folding results from active unfolding of kinetically trapped conformations that partition between subsequent folding and misfolding processes. The transient encapsulation of the protein client within the cylinder is considered to be a by-product of this mechanism. There are several experimental challenges associated with differentiating these potential mechanisms of chaperonin action, realizing that the mechanism that applies could be protein-client specific. First, it is ideal to establish conditions that eliminate reversible and irreversible aggregation during quantitative spontaneous folding in buffer to differentiate these mechanisms. Second, if folding could be demonstrated to occur by a single encapsulation event, this is ideal for eliminating the “iterative annealing” mechanism for a particular client protein. To establish conditions of spontaneous, quantitative refolding, where reversible and irreversible aggregation is not occurring, the authors resorted to single molecule intermolecular fluorescence resonance energy transfer (FRET) measurements at low protein client concentrations. The client or substrate protein employed by the authors is a double mutant of the 41 kDa maltose binding protein (DM-MBP), a monomeric protein that folds slowly (t1/2=35 min at 25 °C) but in quantitative yield. The authors showed that GuHCl-denatured DM-MBP labeled with a small molecule FRET donor at position 312 that was mixed with an equal concentration of GuHCl-denatured DM-MBP labeled at
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position 312 with a small molecule FRET acceptor (50 pM each) did not exhibit a crosscorrelation signal during the spontaneous folding reaction monitored by dual color fluorescence cross-correlation single molecule experiments. Spiking in 5 pM of DMMBP double labeled (DL) with a donor fluorophore at position 30 and an acceptor fluorophore at position 312, to simulate a non-covalent DM-MBP dimer, revealed an easily detected cross-correlation signal, indicating that less than 5% of an aggregate could be readily detected. Moreover, the authors also used fluorescence correlation spectroscopy to show that the number of DM-MBP acceptor labeled fluorophores (at position 312) remained constant over the folding period. This signal would be predicted to change with time if reversible or irreversible aggregation had occurred, providing additional strong evidence for the lack of aggregation during spontaneous folding.
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To facilitate folding in buffer vs. chaperonin-mediated folding rate comparisons, the authors utilized a single pair FRET approach (spFRET)–a single molecule approach. The DM-MBP(DL) FRET chaperonin client introduced above was used for these experiments. The spontaneous folding reaction was stopped at time points of interest by adding GroEL in the absence of ATP, which binds unfolded and misfolded DMMBP(DL), arresting additional folding by serving as a “holdase chaperone”. The natively folded ensemble of conformations exhibit a FRET efficiency of 0.72, whereas 40% of unfolded DM-MBP(DL) exhibits a FRET efficiency of 0.06, consistent with an expanded conformational ensemble. Quantification of the 0.06 and 0.72 FRET efficiencies enabled the authors to extract folding rates at a concentration of 100 pM DM-MBP(DL). The same approach was used to measure chaperonin-assisted DM-MBP(DL) folding rates, except that apyrase was added to deplete the folding reaction of ATP and to convert the chaperonin to a “holdase chaperone”, preventing additional folding during the measurement period. Notably, chaperonin-assisted folding was 5.6 fold faster than spontaneous DM-MBP(DL) folding. Single molecule fluorescence correlation spectroscopy revealed an analogous acceleration in chaperonin assisted folding. Ensemble FRET measurements at the same concentration revealed a 7.7 fold increase in the rate of chaperonin-assisted folding. When position 312 of DM-MBP was labeled only with the fluorescence FRET acceptor Atto655, this electronically excited fluorophore can transfer an electron to tryptophan side chains upon contact, which occurs in the unfolded ensemble. Photoinduced electron transfer coupled with fluorescence correlation spectroscopy was used by the authors to quantify the entropy in the dynamic folding intermediate of DM-MBP in the spontaneous folding reaction and in chaperonin-assisted folding. While binding of DM-MBP to GroEL restricts chain dynamics modestly, GroEL/ES encapsulation results in a marked restriction of chain flexibility, as reflected by the relaxation time of the DM-MBP folding intermediate going from 40 to 99 s. Thus the chemical environment of the chaperonin reduces chain entropy, which is one strategy to reduce the activation energy to get to the native state. That a reduction in chain entropy is key is supported by a previous publication by the Hartl/Hayer-Hartl lab demonstrating that strategic placement of disulfide bonds in MBP renders it a fast folder2.
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To scrutinize the “iterative annealing” model of chaperonin function, several experiments were performed, including employing a single ring GroEL/ES chaperonin variant to demonstrate that a single round of encapsulation inside the chaperonin cylinder is sufficient to achieve hastened DM-MBP folding in quantitative yield. Moreover, dramatically increasing the concentration of native GroEL/ES in the single molecule experiments relative to ensemble experiments did not slow folding kinetics of DM-MBP or decrease yield, which is best rationalized and likely can only be rationalized by encapsulation as the basis of folding catalysis. Under the conditions of the single molecule experiments, the authors present a logical argument that 80% of unfolded or misfolded DM-MBP is always encapsulated, while the remainder is bound to the apical domain of GroEL. Taken together, this is compelling evidence that folding occurs almost exclusively within GroEL/ES, if not entirely there.
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Additional striking evidence that folding and folding catalysis occurs by chaperoninmediated encapsulation of client proteins arises from converting GroEL, which has 42 negative charges on the wall of the cis cavity, to a chaperonin with a net charge of 0. This was accomplished by converting Glu252, Asp253, Glu255, Asp359, Asp361 and Glu363 to Lys residues through mutagenesis. Notably, this KKKKKK-GroEL ceases to be a folding catalyst for DM-MBP, and the photoinduced electron transfer coupled with fluorescence correlation spectroscopy no longer demonstrates that the mobility of the DM-MBP folding intermediate is reduced upon encapsulation. Importantly, the removal of the net negative charge converts the active GroEL/ES chaperonin to a passive chaperonin for the DM-MBP client protein.
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Both the Hartl/Hayer-Hartl lab3 and independently later the Rye lab4 published that RuBisCO refolding is also accelerated by GroEL/ES encapsulation, using a range of single molecule and ensemble measurements. Together they used single molecule experiments and related experiments to demonstrate beyond any reasonable doubt that reversible aggregation of RuBisCO does not occur under the conditions used to demonstrate chaperonin-mediated catalysis of RuBisCO folding. The Hartl/Hayer-Hartl lab also demonstrated recently that GroEL/ES also accelerates the folding of the tetrameric TIM-barrel protein DapA, an enzyme that catalyzes the formation of dihydropicolinic acid. GroEL/ES accelerates folding of DapA 30 fold and renders formation of the folded ensemble more segmental and less cooperative inside the cage relative to spontaneous DapA folding, as discerned by HD exchange experiments5. This mechanism lowers the high entropic component of the folding energy barrier associated with spontaneous folding. In summary, there is now compelling evidence from the manuscripts outlined here that chaperonins can act as an “active cage” for proteins that get stuck in a kinetic trap(s) during spontaneous folding. Interestingly, GroEL/ES functions as an active cage for DMMBP and as a passive cage for wild type MBP assisted folding. All of the data reported to date are consistent with a model for chaperonin-mediated folding catalysis wherein the chemical environment of the chaperonin reduces chain entropy destabilizing kinetically trapped folding intermediates, thus reducing the activation energy required to get to the
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Gupta, A. J., Haldar, S., Milicic, G., Hartl, F. U. & Hayer-Hartl, M. (2014). Active cage mechanism of chaperonin-assisted protein folding demonstrated at single molecule level. Journal of Molecular Biology 426, DOI: 10.1016/j.jmb.2014.04.018. Chakraborty, K., Chatila, M., Sinha, J., Shi, Q., Poschner, B. C., Sikor, M., Jiang, G., Lamb, D. C., Hartl, F. U. & Hayer-Hartl, M. (2010). Chaperonin-catalyzed rescue of kinetically trapped states in protein folding. Cell 142, 112-22. Brinker, A., Pfeifer, G., Kerner, M. J., Naylor, D. J., Hartl, F. U. & Hayer-Hartl, M. (2001). Dual function of protein confinement in chaperonin-assisted protein folding. Cell 107, 223-33. Lin, Z. & Rye, H. S. (2004). Expansion and compression of a protein folding intermediate by GroEL. Mol Cell 16, 23-34. Georgescauld, F., Popova, K., Gupta, A. J., Bracher, A., Engen, J. R., HayerHartl, M. & Hartl, F. U. (2014). GroEL/ES Chaperonin Modulates the Mechanism and Accelerates the Rate of TIM-Barrel Domain Folding. Cell 157, 922-34.
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native state, while preventing aggregation by folding within the cage or cylinder (Figure 1B)1; 2; 3; 4; 5.
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Figure 1
