Partners is innate: no extraneous factors are necessary to elicit this

Partners is innate: no extraneous factors are necessary to elicit this effect in vitro. This finding agrees with an earlier observation that theFigure 4. Interaction of MBP fusion MedChemExpress Pentagastrin proteins with GroEL/S. (A) Lysed cells co-expressing H6-MBP-GFP and either wild-type GroE or the GroE3? variant are shown under blue or white light illumination. Cells co-expressing GroE3? fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the MBP-GFP fusion protein are shown on the left. (B) SDS-PAGE analysis of total and CB5083 soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein. doi:10.1371/journal.pone.0049589.gThe Mechanism of Solubility Enhancement by MBPFigure 5. The addition of GroEL and GroES increases the yield of properly folded passenger proteins in vitro. (A) G3PDH activity. (B) DHFR activity. doi:10.1371/journal.pone.0049589.grecovery of soluble procapthepsin D and pepsinogen after refolding could be enhanced by fusing them to MBP [37], and confirms the (-)-Calyculin A generality of this result. Exactly why MBP is such an effective solubility enhancer (in contrast to many other highly soluble proteins) remains uncertain, but the fact that it can perform this feat in vitro appears to rule out the “chaperone magnet” model. Consistent with an earlier report [38], the experiments described here support a role for the chaperonin GroEL/S in the folding of some passenger proteins but not in solubility enhancement by MBP. Rather, our results indicate that chaperones and/or chaperonins seem to come into play after a passenger protein has been rendered soluble by MBP. Kapust and Waugh suggested that MBP functions as a kind of passive chaperone in the context of a fusion protein [4]. Iterative cycles of binding and release by MBP of 68181-17-9 partially folded passenger proteins eventually results in their spontaneous folding while avoiding the kinetically competing self-aggregation pathway. The hydrophobic ligand-binding pocket in MBP, which is not present in other highly soluble proteins that do not function as solubility enhancers (e.g., GST), was proposed to be the locus of polypeptide binding. The phenotypes of some mutations in MBP were observed to be consistent with this model [25]. However, one might then expect that the occupation of this pocket by maltose, which results in the transition from an “open” to a “closed” complex [39], would impede solubility enhancement by MBP. Yet, at odds with this prediction, we found that the inclusion of as much as 30 mM maltose in refolding experiments did not appreciably reduce the recovery of soluble MBP fusion proteins (MBP has a KD of 1200 nM for maltose [40]). This does not necessarily rule out the intramolecular chaperone model, however, because the proposed interaction site may lie elsewhere on the surface of MBP [8].Two Pathways for the Folding of Passenger ProteinsWe have shown that there are at least two pathways to the native state for passenger proteins that have been rendered soluble by fusing them to MBP. Some proteins such as TEV protease andGFP can fold spontaneously if their propensity to form insoluble aggregates is blocked by fusing them to MBP. Other passenger proteins, exemplified by G3PDH and DHFR, depend on endogenous GroES/L to fold correctly after being solubilized by MBP. In both cases, MBP serves as a kind of “holdase” to maintain the passenger proteins in an aggregation-resistant form th.Partners is innate: no extraneous factors are necessary to elicit this effect in vitro. This finding agrees with an earlier observation that theFigure 4. Interaction of MBP fusion proteins with GroEL/S. (A) Lysed cells co-expressing H6-MBP-GFP and either wild-type GroE or the GroE3? variant are shown under blue or white light illumination. Cells co-expressing GroE3? fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the MBP-GFP fusion protein are shown on the left. (B) SDS-PAGE analysis of total and soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein. doi:10.1371/journal.pone.0049589.gThe Mechanism of Solubility Enhancement by MBPFigure 5. The addition of GroEL and GroES increases the yield of properly folded passenger proteins in vitro. (A) G3PDH activity. (B) DHFR activity. doi:10.1371/journal.pone.0049589.grecovery of soluble procapthepsin D and pepsinogen after refolding could be enhanced by fusing them to MBP [37], and confirms the generality of this result. Exactly why MBP is such an effective solubility enhancer (in contrast to many other highly soluble proteins) remains uncertain, but the fact that it can perform this feat in vitro appears to rule out the “chaperone magnet” model. Consistent with an earlier report [38], the experiments described here support a role for the chaperonin GroEL/S in the folding of some passenger proteins but not in solubility enhancement by MBP. Rather, our results indicate that chaperones and/or chaperonins seem to come into play after a passenger protein has been rendered soluble by MBP. Kapust and Waugh suggested that MBP functions as a kind of passive chaperone in the context of a fusion protein [4]. Iterative cycles of binding and release by MBP of partially folded passenger proteins eventually results in their spontaneous folding while avoiding the kinetically competing self-aggregation pathway. The hydrophobic ligand-binding pocket in MBP, which is not present in other highly soluble proteins that do not function as solubility enhancers (e.g., GST), was proposed to be the locus of polypeptide binding. The phenotypes of some mutations in MBP were observed to be consistent with this model [25]. However, one might then expect that the occupation of this pocket by maltose, which results in the transition from an “open” to a “closed” complex [39], would impede solubility enhancement by MBP. Yet, at odds with this prediction, we found that the inclusion of as much as 30 mM maltose in refolding experiments did not appreciably reduce the recovery of soluble MBP fusion proteins (MBP has a KD of 1200 nM for maltose [40]). This does not necessarily rule out the intramolecular chaperone model, however, because the proposed interaction site may lie elsewhere on the surface of MBP [8].Two Pathways for the Folding of Passenger ProteinsWe have shown that there are at least two pathways to the native state for passenger proteins that have been rendered soluble by fusing them to MBP. Some proteins such as TEV protease andGFP can fold spontaneously if their propensity to form insoluble aggregates is blocked by fusing them to MBP. Other passenger proteins, exemplified by G3PDH and DHFR, depend on endogenous GroES/L to fold correctly after being solubilized by MBP. In both cases, MBP serves as a kind of “holdase” to maintain the passenger proteins in an aggregation-resistant form th.Partners is innate: no extraneous factors are necessary to elicit this effect in vitro. This finding agrees with an earlier observation that theFigure 4. Interaction of MBP fusion proteins with GroEL/S. (A) Lysed cells co-expressing H6-MBP-GFP and either wild-type GroE or the GroE3? variant are shown under blue or white light illumination. Cells co-expressing GroE3? fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the MBP-GFP fusion protein are shown on the left. (B) SDS-PAGE analysis of total and soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein. doi:10.1371/journal.pone.0049589.gThe Mechanism of Solubility Enhancement by MBPFigure 5. The addition of GroEL and GroES increases the yield of properly folded passenger proteins in vitro. (A) G3PDH activity. (B) DHFR activity. doi:10.1371/journal.pone.0049589.grecovery of soluble procapthepsin D and pepsinogen after refolding could be enhanced by fusing them to MBP [37], and confirms the generality of this result. Exactly why MBP is such an effective solubility enhancer (in contrast to many other highly soluble proteins) remains uncertain, but the fact that it can perform this feat in vitro appears to rule out the “chaperone magnet” model. Consistent with an earlier report [38], the experiments described here support a role for the chaperonin GroEL/S in the folding of some passenger proteins but not in solubility enhancement by MBP. Rather, our results indicate that chaperones and/or chaperonins seem to come into play after a passenger protein has been rendered soluble by MBP. Kapust and Waugh suggested that MBP functions as a kind of passive chaperone in the context of a fusion protein [4]. Iterative cycles of binding and release by MBP of partially folded passenger proteins eventually results in their spontaneous folding while avoiding the kinetically competing self-aggregation pathway. The hydrophobic ligand-binding pocket in MBP, which is not present in other highly soluble proteins that do not function as solubility enhancers (e.g., GST), was proposed to be the locus of polypeptide binding. The phenotypes of some mutations in MBP were observed to be consistent with this model [25]. However, one might then expect that the occupation of this pocket by maltose, which results in the transition from an “open” to a “closed” complex [39], would impede solubility enhancement by MBP. Yet, at odds with this prediction, we found that the inclusion of as much as 30 mM maltose in refolding experiments did not appreciably reduce the recovery of soluble MBP fusion proteins (MBP has a KD of 1200 nM for maltose [40]). This does not necessarily rule out the intramolecular chaperone model, however, because the proposed interaction site may lie elsewhere on the surface of MBP [8].Two Pathways for the Folding of Passenger ProteinsWe have shown that there are at least two pathways to the native state for passenger proteins that have been rendered soluble by fusing them to MBP. Some proteins such as TEV protease andGFP can fold spontaneously if their propensity to form insoluble aggregates is blocked by fusing them to MBP. Other passenger proteins, exemplified by G3PDH and DHFR, depend on endogenous GroES/L to fold correctly after being solubilized by MBP. In both cases, MBP serves as a kind of “holdase” to maintain the passenger proteins in an aggregation-resistant form th.Partners is innate: no extraneous factors are necessary to elicit this effect in vitro. This finding agrees with an earlier observation that theFigure 4. Interaction of MBP fusion proteins with GroEL/S. (A) Lysed cells co-expressing H6-MBP-GFP and either wild-type GroE or the GroE3? variant are shown under blue or white light illumination. Cells co-expressing GroE3? fluoresce more intensely than cells co-expressing wild-type GroE as a result of enhanced GFP folding. Cells expressing only the MBP-GFP fusion protein are shown on the left. (B) SDS-PAGE analysis of total and soluble proteins from the cells in (A). T, total intracellular protein; S, soluble intracellular protein. doi:10.1371/journal.pone.0049589.gThe Mechanism of Solubility Enhancement by MBPFigure 5. The addition of GroEL and GroES increases the yield of properly folded passenger proteins in vitro. (A) G3PDH activity. (B) DHFR activity. doi:10.1371/journal.pone.0049589.grecovery of soluble procapthepsin D and pepsinogen after refolding could be enhanced by fusing them to MBP [37], and confirms the generality of this result. Exactly why MBP is such an effective solubility enhancer (in contrast to many other highly soluble proteins) remains uncertain, but the fact that it can perform this feat in vitro appears to rule out the “chaperone magnet” model. Consistent with an earlier report [38], the experiments described here support a role for the chaperonin GroEL/S in the folding of some passenger proteins but not in solubility enhancement by MBP. Rather, our results indicate that chaperones and/or chaperonins seem to come into play after a passenger protein has been rendered soluble by MBP. Kapust and Waugh suggested that MBP functions as a kind of passive chaperone in the context of a fusion protein [4]. Iterative cycles of binding and release by MBP of partially folded passenger proteins eventually results in their spontaneous folding while avoiding the kinetically competing self-aggregation pathway. The hydrophobic ligand-binding pocket in MBP, which is not present in other highly soluble proteins that do not function as solubility enhancers (e.g., GST), was proposed to be the locus of polypeptide binding. The phenotypes of some mutations in MBP were observed to be consistent with this model [25]. However, one might then expect that the occupation of this pocket by maltose, which results in the transition from an “open” to a “closed” complex [39], would impede solubility enhancement by MBP. Yet, at odds with this prediction, we found that the inclusion of as much as 30 mM maltose in refolding experiments did not appreciably reduce the recovery of soluble MBP fusion proteins (MBP has a KD of 1200 nM for maltose [40]). This does not necessarily rule out the intramolecular chaperone model, however, because the proposed interaction site may lie elsewhere on the surface of MBP [8].Two Pathways for the Folding of Passenger ProteinsWe have shown that there are at least two pathways to the native state for passenger proteins that have been rendered soluble by fusing them to MBP. Some proteins such as TEV protease andGFP can fold spontaneously if their propensity to form insoluble aggregates is blocked by fusing them to MBP. Other passenger proteins, exemplified by G3PDH and DHFR, depend on endogenous GroES/L to fold correctly after being solubilized by MBP. In both cases, MBP serves as a kind of “holdase” to maintain the passenger proteins in an aggregation-resistant form th.