Productive and Nonproductive Intermediates in the Folding of Denatured Rhodanese

Abstract

The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 m urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species. The competition between protein aggregation and folding has been investigated using rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) as a model. During folding from a urea-denatured state, rhodanese rapidly forms associated species or intermediates, some of which are large and/or sticky. The early removal of such particles by filtration results in a decreased refolding yield. With time, a portion of the smaller aggregates can partition back first to intermediates and then to refolded protein, while a fraction of these irreversibly form unproductive higher aggregates. Dynamic light scattering measurements indicate that the average sizes of the aggregates formed during rhodanese folding increase from 225 to 325 nm over 45 min and they become increasingly heterogeneous. Glycerol addition or the application of high hydrostatic pressure improved the final refolding yields by stabilizing smaller particles. Although addition of glycerol into the refolding mixture blocks the formation of unproductive aggregates, it cannot dissociate them back to productive intermediates. The presence of 3.9 m urea keeps the aggregates small, and they can be dissociated to monomers by high hydrostatic pressure even after 1 h of incubation. These studies suggest that early associated intermediates formed during folding can be reversed to give active species. 2-mercaptoethanol The renaturation of a denatured protein typically involves a number of intermediate states (on-pathway or off-pathway) leading to either correctly folded or misfolded structures (1Baldwin R.L. Fold. Des. 1995; 1: R1-R8Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 2Hagihara Y. Goto Y. Fink A.L. Goto Y. Molecular Chaperones in the Life Cycle of Proteins. Marcel Dekker, New York1998: 1-33Google Scholar). Protein aggregation is one of the main side reactions during refolding, and it often results from interactions among partially folded intermediates (3Jaenicke R. Seckler R. Wetzel R. Advances in Protein Chemistry. Academic Press, New York1997: 1-59Google Scholar). In general, this aggregation, although ubiquitous and important for basic and applied problems, is difficult to study. The enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) is an interesting model for studying issues related to such problems in protein folding (4Mendoza J.A. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 16973-16976Abstract Full Text PDF PubMed Google Scholar, 5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar, 6Horowitz P.M. Butler M. J. Biol. Chem. 1993; 268: 2500-2504Abstract Full Text PDF PubMed Google Scholar, 7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar, 8Gorovits B.M. Horowitz P.M. Biochemistry. 1998; 37: 6132-6135Crossref PubMed Scopus (94) Google Scholar). This monomeric protein containing 293 amino acids (mass = 33 kDa) is folded into two independent, equal-size domains, and its crystal structure is available (9Ploegman J.H. Drent G. Kalk K.H. Hol W.G. Heinrikson R.L. Keim P. Weng L. Russell J. Nature. 1978; 273: 124-129Crossref PubMed Scopus (214) Google Scholar). The domains are tightly associated and the interdomain surface is highly hydrophobic. Rhodanese contains four cysteine residues, Cys-63, Cys-247, Cys-254, and Cys-263, which are all reduced in the active protein (9Ploegman J.H. Drent G. Kalk K.H. Hol W.G. Heinrikson R.L. Keim P. Weng L. Russell J. Nature. 1978; 273: 124-129Crossref PubMed Scopus (214) Google Scholar). During unfolding/refolding in the presence of denaturants, disulfides can form among these cysteines, leading to misfolded structures in addition to aggregation. The disulfide-containing species leading to misfolding have been characterized (10Horowitz P.M. Hua S. Biochim. Biophys. Acta. 1995; 1249: 161-167Crossref PubMed Scopus (10) Google Scholar) with SDS-polyacrylamide gel electrophoresis, and the formation of aggregates during unfolding has been reported earlier (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar, 11Tandon S. Horowitz P.M. J. Biol. Chem. 1989; 264: 9859-9866Abstract Full Text PDF PubMed Google Scholar). It has been established that during unfolding, the presence of reductants such as β-ME1 and thiosulfate prevent the formation of most of the disulfides and better refolding yields are observed (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar). However, refolding at low temperature (e.g. 12 °C), even in the presence of 200 mmβ-ME, 50 mm thiosulfate, and at very low protein concentration (e.g. 3.6 μg/ml), led to incomplete recovery of activity (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar, 11Tandon S. Horowitz P.M. J. Biol. Chem. 1989; 264: 9859-9866Abstract Full Text PDF PubMed Google Scholar). Folding intermediates formed during chemical or thermal denaturation often contain similar levels of secondary structure as the native proteins, but they contain a decreased number of tertiary contacts (12Ptitsyn O.B. Creighton T.E. Protein Folding. W. H. Freeman and Co., New York1992: 243-300Google Scholar), partially or completely dissociated domains (13Horowitz P.M. Criscimagna N.L. J. Biol. Chem. 1990; 265: 2576-2583Abstract Full Text PDF PubMed Google Scholar), and/or incorrectly formed disulfide bonds (14Ewbank J.J. Creighton T.E. Nature. 1991; 350: 518-520Crossref PubMed Scopus (134) Google Scholar). As a result, such intermediates tend to be highly hydrophobic, and consequently they can easily form large aggregates and precipitate. It was suggested that, at intermediate concentrations of denaturant, the two domains of rhodanese dissociate (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. 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The stabilizing of some have been partially to decreased which protein from with during the in which they are to Biochemistry. 1990; PubMed Scopus Google Scholar) with the of in Molecular Press, Scholar). have that high hydrostatic can dissociate protein oligomers G. Biochemistry. PubMed Scopus Google Scholar, G. Protein and Inc., New Scholar, in Molecular and Press, New Google Scholar, Biochemistry. 1998; 37: PubMed Scopus Google Scholar). In high hydrostatic pressure has been to dissociate protein aggregates formed in vitro B.M. Horowitz P.M. Biochemistry. 1998; 37: 6132-6135Crossref PubMed Scopus (94) Google Scholar), and to an increase in the folding by intermediates to In the have investigated the and of aggregates formed during rhodanese folding the of the of these a for rhodanese folding is The the that dissociated species can be and into a productive folding rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was as described and was at as a in containing 1 mm thiosulfate R. Horowitz P.M. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). the a rhodanese from was by gel filtration using a and with m = Rhodanese concentrations using a of = Chem. Google Scholar). In the for refolding and filtration of aggregates at low protein concentrations μg/ml), it was to by using the or In these a was using the of rhodanese from folding was by the in The at the which was at nm for all are in the for The was to with a and of by measurements of protein after denaturation in the measurements that from in the misfolded intermediates the and species in the In the it was to the of species to the was from of containing glycerol and rhodanese glycerol containing 200 mm 50 mm thiosulfate, and 50 mm was this (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar). containing glycerol by from a m of glycerol to the and then the final of the by of the Rhodanese activity was by a at the formation of the between and one of the Chem. Google Scholar). of of the enzyme to 1 of mixture and for The mixture was at = and of of thiosulfate, m and The was by of was by of The presence of of or glycerol after of the enzyme in the mixture with the The of the refolded rhodanese with to the activity of a of native enzyme rhodanese was at by of native enzyme into m 50 mm 50 mm thiosulfate, 1 mm = and for at one (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. 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PubMed Scopus Google Scholar). rhodanese was into a containing 50 mm 200 mm 50 mm thiosulfate, a final urea concentration of 3.9 m and a final protein concentration of of the to between and The of the light was at using light at rhodanese refolding high hydrostatic to at min after of the denatured These for and the as described The activity of the native protein at pressure was as Rhodanese was by as described B.M. J. Horowitz P.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). protein at a final concentration of was in was from a in to a final concentration of The mixture was for h at temperature and then containing m to the and rhodanese was to protein aggregation Rhodanese with was into the containing 3.9 m In some rhodanese was to the final protein concentration at the as described The of the was using an at and the was detected at as in 1 The to either or = 1 1 or 1 1 The was the of activity at The 1 and and the from of the using the Rhodanese at was in m urea and then to give a final urea concentration of mm and m glycerol to rhodanese to The refolding of rhodanese is in the presence of glycerol (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar) of the of the process by the The of these kinetic from the to the yields in these of the activity be with a of min at = 3.6 The in 1 that the final decreased as the rhodanese concentration as reported earlier (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar). The observed from = and for and 50 The yields and at and 50 glycerol was in the The concentration of the observed and the yields from the in 1 are in and Such of the and of activity are with results (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar). These the for folding and aggregation at protein concentrations from the rhodanese in to to in states of In this the urea concentrations in the final refolding the This in urea from to m in the final refolding in the enzyme activity of glycerol was or using a in which the final with urea that the rhodanese concentrations refolded at the final urea concentration of m These results that the results of The of the aggregates that formed during the rhodanese folding by light scattering measurements at after refolding, as described The results are in where the the at the the have been the the of the the and of particles the is the of the of the and of the 1991; PubMed Scopus (10) Google Scholar). The average of particles at the of the and the average at of the a of the to the of this Biochemistry. 1990; PubMed Scopus Google Scholar, 1991; PubMed Scopus (10) Google Scholar). the first after the in that particles with average of nm can be detected of native rhodanese is The of the light the of 1 that the particles observed formed The average of the particles from nm to nm over min The of the during this as by the increase of at of the from nm at 1 min to nm at The of intermediates or particles formed during refolding by a in which refolding was at a high concentration for refolding was the protein into at 50 of the refolding rhodanese to a final concentration of 3.6 and then that, after the refolding was the protein of the and dissociate the aggregates that formed at that The of activity to The to a and two for the and The observed = to a of the = to a of The important from this is that the a to activity in min after which is a of activity have the but it be to of by the formation of misfolded structures from of the intermediates. This the of intermediates that to the of activity to the formation of species. the species for the aggregates formed of high enzyme then they have dissociated in of at these final = 3.6 In that a of of activity have been a at of the observed in of refolding enzyme at of rhodanese was in to at 50 and then the refolding mixture was to 3.6 of protein at with the m The after the final the is from to a as described The and the the and that using the from the of the of the particles formed of denatured rhodanese be a a process between and The results are in the early in the refolding process filtration at the renaturation of the protein in the was at the with an and the activity with to give a final that was decreased to of have been observed in the of a portion of the protein that have in was the the and the at 45 However, a portion of the protein that the was active but it to as two forms of protein that in a one was the and the the was observed for a min after renaturation was a smaller increase in activity from after filtration to a recovery of a the the was after min of refolding the final concentration of the active enzyme was very to that in the It was from the reduced final yields that filtration species that able to to the of The increase in activity in the that some species the and become active with The results in that the of protein in the rapidly over the first min and then with the that reduced recovery of activity after filtration with the removal of protein with of species formed during incubation. It be that, in of native or urea denatured rhodanese the these very large of protein refolding rhodanese was to the of aggregates that formed after the intermediates Formation of aggregates large to light was using a with and at The results are in The kinetic to aggregation formed at = in that the presence of m glycerol at = the aggregation by this The kinetic the results of as rhodanese was refolded in glycerol at and after to by an of containing m glycerol that the final 50 mm 50 200 mm = and glycerol for for such glycerol after refolding the protein in for 1 min min and min The at the are to a of and of and The from these is that the of large aggregates that form in the are reversed to productive smaller even by the addition of However, the formation of such aggregates can be in the very early by the presence of Rhodanese aggregates the m urea in refolding B.M. Horowitz P.M. Biochemistry. 1998; 37: 6132-6135Crossref PubMed Scopus (94) Google Scholar). This by the of the has a of scattering measurements of these in the presence of urea the of particles with a of nm after for these in formation of particles with of nm although the protein concentration was higher in the light scattering in μg/ml), the presence of urea led to a reduced but it Rhodanese aggregation was reversed the was to In this aggregation was to for and the pressure was to the of the light was reduced to the of This that the aggregation was to hydrostatic pressure was able to the aggregation. These results where the of large aggregates by pressure was B.M. Horowitz P.M. Biochemistry. 1998; 37: 6132-6135Crossref PubMed Scopus (94) Google Scholar) but its aggregates was have a better of the of the protein high hydrostatic rhodanese was as a Rhodanese was by and it was with protein aggregation. of the was to the of the protein aggregates The B.M. J. Horowitz P.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) of of the formation of large B.M. J. Horowitz P.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the protein at 3.9 m urea and pressure in a increase in the of with a of an Protein aggregation these forms large particles that the a process that is in the increase of the the was to after for the decreased to the of the aggregates into monomers the pressure in an increase in The of the that was observed high pressure is very to that observed for the of rhodanese in the of urea In a the of native rhodanese was as a of pressure in the of in the was detected pressure from 1 to These measurements are to by light the is to the formation of while the light scattering is by the species. using the observed and the of rhodanese suggest that the enzyme as a in protein aggregates formed in the presence of 3.9 m urea to be completely dissociated the protein is to The of high hydrostatic pressure to dissociate protein aggregates that pressure can be to the among protein aggregates formed during rhodanese a of rhodanese was min after from the and refolding was to high the final of the native enzyme was containing This can be by the of high pressure to formation of large aggregates. in the large aggregates after are after time, high pressure is in the rhodanese is for min the final of activity is which is to that observed for enzyme that was refolded at 1 the pressure was applied min after the of the final activity a that was that observed for the refolded at 1 In the of the yields of refolding and at and 1 These yields at and at pressure are with earlier results in m refolding B.M. Horowitz P.M. Biochemistry. 1998; 37: 6132-6135Crossref PubMed Scopus (94) Google Scholar), although in studies the of pressure at refolding and the of activity was These results that even pressure dissociated the aggregates the activity be to a the refolding mixture was at early but at very of the aggregates as a of of rhodanese after the refolding for activity at at the in a rhodanese contains domains that are tightly associated at an containing a number of As rhodanese it forms at one intermediate that (6Horowitz P.M. Butler M. J. Biol. Chem. 1993; 268: 2500-2504Abstract Full Text PDF PubMed Google Scholar). During formation of these states the unfolding between and m urea = can be detected in its secondary (13Horowitz P.M. Criscimagna N.L. J. Biol. Chem. 1990; 265: 2576-2583Abstract Full Text PDF PubMed Google Scholar) and tertiary (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar) = m the concentration of urea mm = 50 mm thiosulfate, and 200 mm at which of rhodanese activity is observed (6Horowitz P.M. Butler M. J. Biol. Chem. 1993; 268: 2500-2504Abstract Full Text PDF PubMed Google Scholar). In these intermediates, it has been suggested that the domains but dissociated (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar). As a result, the protein is able to form which to aggregation. In addition to aggregation, problems such as disulfide formation and misfolding to its for of protein folding of folding intermediates between productive folding and formation of aggregates (3Jaenicke R. Seckler R. Wetzel R. Advances in Protein Chemistry. Academic Press, New York1997: 1-59Google Scholar, Biochemistry. 1990; PubMed Scopus Google Scholar, Rudolph R. Jaenicke R. Biochemistry. 1991; PubMed Scopus Google Scholar, Y. Protein PubMed Scopus Google Scholar, R. Biol. Chem. 1998; Google Scholar). The rhodanese refolding has been partially described earlier (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar), and it is and the of results in with the described in 7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar). In this is an state, are intermediates, the native state, and 1 are intermediate species leading to the formation of large aggregates. folding by of the denatured rhodanese is suggested to to form S. Horowitz P.M. J. Biol. Chem. 1989; 264: 9859-9866Abstract Full Text PDF PubMed Google Scholar), which rapidly to the forms and is able to to the active while a to the and then in to form active is to aggregation that as the aggregates become The concentration and high of the aggregation in the of refolding at high protein concentrations (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar, 7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar). This is in the concentration of the refolding 1 and of that reported in the (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar) and presence of glycerol (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar). As suggested (7Gorovits B.M. McGee W.A. Horowitz P.M. Biochim. Biophys. Acta. 1998; 1382: 120-128Crossref PubMed Scopus (31) Google Scholar), these to the the recovery is reduced at high protein large the folding the This the that, as the protein concentration the refolding but the folding This is similar to that for refolding of in the presence of Y. Protein PubMed Scopus Google Scholar). In addition to aggregation, of the intermediates can misfolding (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar, P.M. Hua S. Biochim. Biophys. Acta. 1995; 1249: 161-167Crossref PubMed Scopus (10) Google Scholar) as in even glycerol aggregation to of the activity be recovery of activity refolding has been reported for and in a similar and it was that the refolding from m of its activity in and in m glycerol Full Text PDF PubMed Scopus Google Scholar). The filtration that some aggregates can be the As from and the concentration of protein the active enzyme in the to increase and was folding in the This that the smaller productive intermediates, which the In the reduced levels of recovery after filtration at early that all of the protein the is of unproductive large aggregates, but it contains species that can produce active enzyme in of 1 and of to the and in to form active protein can for the of rhodanese refolding observed in the The between which is very and which is can be in m the formation of large can be However, glycerol was after the refolding to some the formation of large aggregates but the that formed dissociate in hydrostatic pressure is to dissociate protein oligomers and aggregates, and the results are with the that high hydrostatic pressure can and protein aggregates G. Protein and Inc., New Scholar, in Molecular and Press, New Google Scholar). the rhodanese was after the renaturation was the final refolding was This can be by the of pressure to the between the and 1 species formation of monomeric folding intermediate As a result, the of refolded enzyme can be In high concentrations of glycerol prevent the formation of large unproductive aggregates, and high hydrostatic pressure the aggregates that are the of rhodanese In results from this a for the reported (5Mendoza J.A. Rogers E. Lorimer G.H. Horowitz P.M. J. Biol. Chem. 1991; 266: 13587-13591Abstract Full Text PDF PubMed Google Scholar) that rhodanese refolding is the denatured enzyme is to urea concentrations to the unfolding native with to very low urea concentrations of urea to intermediates that can to folding and prevent formation of large aggregates. These results that aggregation can and aggregates can be to give active protein, is applied early in the aggregation It is that this in the folding of that are to aggregation, and in to aggregation in the recovery of active