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Nucleation in protein folding-confusion of structure and process
TIBS 15-NOVEMBER 1990
TALKINGPOINT DESPITE THE APPEARANCE of substantial evidence that the mechanism by which protein folding occurs does not correspond to a nucleation mechanism in the kinetic sense L~, many investigators continue to discuss nucleation sites and nuclei in relation to protein folding~ . This paradox arises from the fact that 'nucleation', as currently used, refers both to the kinetics of folding and to the formation of bits of structure early in the folding process. The initial formation of small, condensed regions of organized structures is a feature of many of the current models of folding 3,4,7-0.A rich variety of names has been given to these marginally stable bits of structure. In addition to 'nuclei '1°-~2, the structures have been called LINCS~3, condensed microdomains TM, clusters ~s, islands ~6, fluctuating secondary structures ~7, local structures TM, fluctuating embryos ~°, kernels ~, foldons2°, seeds 2~ and chainfolding initiation sites s. Yet another name given to this structural construct, flickering clusters 22, is a term adopted from earlier work in the field of water structure 23. Of course no claim of completeness is made for this list. Some of the above are not quite synonymous but denote some specific aspect of structure. For example, 'fluctuating secondary structures' implies that peptide-peptide hydrogen bonding is central to the marginal stability of these primitive structures, while the stabilization of 'clusters' is presumed to arise from structurally more permissive hydrophobic interactions. While these early structures are seen by some to be essentially the same as they are in the native protein (i.e. in their 'native format 'u) strict structural identity is not always envisioned. Notwithstanding the variety in the above terminology, the terms all imply initiating structures in folding. They are therefore structural nuclei. In kinetic nucleation, as established in the crystallization of polyethylene and other synthetic polymers 24, nu-
D. B. Wetlaufer is at the Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
414
....
'
~'i
i( i ii'
The t e r m 'nucleation' is currently used to denote two distinctly different aspects of folding: the kinetic and the structural. This gives rise to ambiguity in the use of the word 'nucleation', which is compounded by the fact that the word 'nuclei', as used in the structural sense, has more aliases than cats have lives.
For the handful of globular proteins cleation is an initial rate-limiting step in folding, which is so much slower than that have been examined, folding does the following steps that no downstream not involve kinetic nucleation, but intermediates accumulate. This kind of appears to proceed with the formation nucleation was described in an early of structural nuclei. For collagen, on the study on collagen folding2s, in which the other hand, folding appears to involve authors formulate a 'scheme involving kinetic nucleation6,25. Baldwin recognized the problem of an intermediate, the formation of which is considered to be rate-limiting'. This dual meanings for nucleation, and statement is equivalent to describing col- proposed different terms for structural lagen folding as a kinetically nucleated nuclei 1,21,22.Unfortunately his proposals process. On the other hand, experimen- have not taken hold. I have experienced tal results on the folding of globular similar difficulties with a proposal to proteins show the existence of sizeable distinguish between 'protein folding' as populations of various intermediates. a process and 'protein folding' as a Hence there is no initial rate-limiting synonym for protein structure 27. step, and these are not kinetically Nevertheless, it is necessary to continue attempting to refine our language. nucleated processes 1,2. The issue of nucleation also arises in Since it is evident that 'nucleation' and the Zimm-Bragg theory of the helix-coil 'nuclei' will persist in folding distransition 26. cussions, it would be useful if these terms were prefaced with 'kinetic' or 'The sharpness of the transition is due to 'structural' to clarify the intended the following consequence of the model. meaning. The formation of the first turn of the helix is [energetically] difficult because References 1 Kim, P. S. and Baldwin,R. L. (1982) Annu. Rev. of a large reduction in entropy. Once Biochem. 51, 459-489 formed, however, this turn acts as a 2 Creighton,T. E. (1990) in Protein Folding nucleus to which further turns can add (Gierasch, L. M. and King, J., eds), pp. by hydrogen bonding. Thus this transform157-170, AmericanAssociationfor the ation has the property of nucleation Advancementof Science characteristic of other sharp transitions.' 3 Wetlaufer, D. B. (1990) in Protein Engineering 'Nucleus' and 'nucleation' in the model of Zimm and Bragg are structural, not kinetic. Their theory, which is very successful in treating high molecular weight homopolypeptides, deals only with the equilibria between helical and random coil forms, and not at all with the kinetics.
(Narang, S,, ed.), pp. 21-35, Butterworths 4 Somorjai, R. L. (1990) in Protein Engineering (Narang, S., ed.), pp. 1-19, Butterworths 5 Montelione,G. T. and Scheraga,H. A. (1989) Acc. Chem. Res. 22, 70-76 6 Brodsky,B. (1990)in Protein Folding (Gierasch, L. M. and King, J., eds), pp. 85-94, American Association for the Advancementof Science 7 Dyson, H. J., Lerner, R. A. and Wright, P. E. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 305-324
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
TIBS 15- NOVEMBER1990 8 Bashford, D., Karplus, M. and Weaver, D. L. (1990) in Protein Folding(Gierasch, L. M. and King, J., eds), pp. 283-290, American Association for the Advancement of Science 9 Goldberg, M. E. (1990) in Protein Folding (Gierasch, L. M. and King, J., eds), pp. 143-154, American Association for the Advancement of Science 10 Tsong, T. Y., Baldwin, R. L. and McPhie, P. (1972) ./. Mol. Biol. 63, 453-475 11 Sachs, D., Schecter, A. N., Eastlake, A. and Anfinsen, C. B. (1972) Prec. Natl Acad. Sci. USA 69, 3790-3794 22 Wetlaufer, D. B. (1973) Prec. Natl Acad. Sci. USA 70, 697-701 13 Rose, G. D., Winter, R. H. and Wetlaufer, D. B. (1976) FEBS Lett. 63, 10-16
MlCROTUBUI.I:-~; are one of the major types of filamentous protein structures of eukaryotic cells. The basic building block of microtubules is tubulin, a heterodimer made up of two related protein subunits, (z and [3 (Ref. 1). The in vitro polymerization of tubulin dimers has been well documented over the past two decades but our understanding of in vivo microtubule assembly remains very limited'. In the intracellular assembly of protein complexes, it is generally assumed that all of the information necessary for the proper folding of polypeptide chains, and their assembly into oligomeric complexes, is contained within the primary sequence of the polypeptide(s) 2. However, this dogma is now being seriously challenged by the discovery of a class of proteins referred to as 'molecular chaperones', which play an essential intermediary role in the correct folding of polypeptide chains, their intra- and extracellular transport, and their assembly into oligomeric protein complexes a,4. Two of the prominent molecular chaperone proteins that are ubiquitous belong to the chaperonin (synonyms: hsp60, GroEL homolog) and hsp70 protein families. The assembly and function of microtubules in vivo has also been tackled in my laboratory by a combined genetic and biochemical approach ~, the rationale and usefulness of which has been discussed elsewhere 7,8. These studies R. S, Gupta is at the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
14 Karplus, M. and Weaver, D. L. (1976) Nature 260, 404-406 15 Kanehisa, M. I. and Tsong, T. Y. (1978) J. Mol. Bio/. 124, 177-194 16 Wako, J. and Sato, N. (1978) J. Phys. Soc. Japan 44, 1939-1945 17 Ptitsyn, O. B. and Finkelstein, A. V. (1980) in Protein Folding (Jaenicke, R., ed.), pp. 101-106, Elsevier 18 Go, N., Abe, H., Mizuno, H. and Taketomi, H. (1980) in Protein Fo/ding(Jaenicke, R., ed.), pp. 167-181, Elsevier 19 Finkelstein, A. V. (1981) in Biomo/ecu/ar Structure, Conformation, Function, and Evolution, VoL 2 (Srinvasan, R., ed.), pp. 103-110, Pergamon Press
20 Yu, M-H. and King, J. (1984) Prec. Nat/Acad. Sci. USA 81, 6584-6588 21 Baldwin, R. L. (1986) Trends Biochem. ScL 11, 6-10 22 Baldwin, R. L. (1989) Trends Biochem. ScL 14, 291-294 23 Frank, H. S. and Wen, W-Y. (1957) Discuss. Faraday Soc. 24, 133-140 24 Gornick, F. and Hoffman, J. D. (1966) in Nucleation Phenomena, pp. 53-65, American Chemical Society 25 Flory, P. J. and Weaver, E. S. (1960) J. Am. Chem. Soc. 82, 4518-4525 26 Zimm, B. H. and Bragg, J. K. (1958) J. Chem. Phys. 28, 1246-1247 27 Wetlaufer, D. B. and Ristow, S. S. (1973) Annu. Rev. Biochem. 42, 135-158
Two proteins, P1 and P2, which are specifically altered in mammalian cell mutants resistant to antimitotic drugs, have been identified as the homologs of two members of the class of proteins known as molecular chaperones. P1 is localized in mitochondria and P2-related proteins are involved in the translocation of proteins to mitochondria. To account for these and a number of other observations, a new model for in vivo microtubule assembly is proposed. reveal that in a number of independent Chinese hamster ovary (CHO) cell mutants selected for resistance to antimitotic drugs, there is an alteration in the electrophoretic properties of two major cellular proteins, designated P1 (~60 kDa) and P2 (-70 kDa)5,6. These proteins have now been identified as homologs of chaperonin and hsp709,'°, respectively. P1 is localized within mitochondria that associate with microtubules in interphase cellsTM. These results raise a number of important questions regarding the role of mitochondria and molecular chaperone proteins in the in vivo assembly of microtubules.
Identification of P1 and P2 as homologsof the chaperonlnand hsp70 proteins Mutants expressing altered P1 and/or P2 exhibit highly specific changes in their resistance and sensitivity towards
© 1990,ElsevierSciencePublishersLtd, (UK) 0376-5067/90/$02.00
other antimitotic drugs, but no cross resistance is observed for any unrelated compounds 6,7. Reduced binding of [3H]podophyllotoxin and [SH]colchicine in cell extracts of these mutants indicate that their mutations either directly or indirectly affect binding of the antimitotic drugs to their cellular target (i.e. tubulin). Furthermore, co-release of P1, P2 and tubulin from cellular fractions under a number of different conditions (incubation at 4°C or with 5 mM CaC12) suggested that these proteins were associated with tubulin/microtubules in vivo 5,6. However, one puzzling observation that was difficult to reconcile with microtubule structure and/or assembly was that P1 and P2 were major cellular proteins, present in approximately equimolar amounts with tubulin. To investigate the cellular distribution and function of P1 and P2, specific antibodies to these proteins were
425
TALKINGPOINT DESPITE THE APPEARANCE of substantial evidence that the mechanism by which protein folding occurs does not correspond to a nucleation mechanism in the kinetic sense L~, many investigators continue to discuss nucleation sites and nuclei in relation to protein folding~ . This paradox arises from the fact that 'nucleation', as currently used, refers both to the kinetics of folding and to the formation of bits of structure early in the folding process. The initial formation of small, condensed regions of organized structures is a feature of many of the current models of folding 3,4,7-0.A rich variety of names has been given to these marginally stable bits of structure. In addition to 'nuclei '1°-~2, the structures have been called LINCS~3, condensed microdomains TM, clusters ~s, islands ~6, fluctuating secondary structures ~7, local structures TM, fluctuating embryos ~°, kernels ~, foldons2°, seeds 2~ and chainfolding initiation sites s. Yet another name given to this structural construct, flickering clusters 22, is a term adopted from earlier work in the field of water structure 23. Of course no claim of completeness is made for this list. Some of the above are not quite synonymous but denote some specific aspect of structure. For example, 'fluctuating secondary structures' implies that peptide-peptide hydrogen bonding is central to the marginal stability of these primitive structures, while the stabilization of 'clusters' is presumed to arise from structurally more permissive hydrophobic interactions. While these early structures are seen by some to be essentially the same as they are in the native protein (i.e. in their 'native format 'u) strict structural identity is not always envisioned. Notwithstanding the variety in the above terminology, the terms all imply initiating structures in folding. They are therefore structural nuclei. In kinetic nucleation, as established in the crystallization of polyethylene and other synthetic polymers 24, nu-
D. B. Wetlaufer is at the Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
414
....
'
~'i
i( i ii'
The t e r m 'nucleation' is currently used to denote two distinctly different aspects of folding: the kinetic and the structural. This gives rise to ambiguity in the use of the word 'nucleation', which is compounded by the fact that the word 'nuclei', as used in the structural sense, has more aliases than cats have lives.
For the handful of globular proteins cleation is an initial rate-limiting step in folding, which is so much slower than that have been examined, folding does the following steps that no downstream not involve kinetic nucleation, but intermediates accumulate. This kind of appears to proceed with the formation nucleation was described in an early of structural nuclei. For collagen, on the study on collagen folding2s, in which the other hand, folding appears to involve authors formulate a 'scheme involving kinetic nucleation6,25. Baldwin recognized the problem of an intermediate, the formation of which is considered to be rate-limiting'. This dual meanings for nucleation, and statement is equivalent to describing col- proposed different terms for structural lagen folding as a kinetically nucleated nuclei 1,21,22.Unfortunately his proposals process. On the other hand, experimen- have not taken hold. I have experienced tal results on the folding of globular similar difficulties with a proposal to proteins show the existence of sizeable distinguish between 'protein folding' as populations of various intermediates. a process and 'protein folding' as a Hence there is no initial rate-limiting synonym for protein structure 27. step, and these are not kinetically Nevertheless, it is necessary to continue attempting to refine our language. nucleated processes 1,2. The issue of nucleation also arises in Since it is evident that 'nucleation' and the Zimm-Bragg theory of the helix-coil 'nuclei' will persist in folding distransition 26. cussions, it would be useful if these terms were prefaced with 'kinetic' or 'The sharpness of the transition is due to 'structural' to clarify the intended the following consequence of the model. meaning. The formation of the first turn of the helix is [energetically] difficult because References 1 Kim, P. S. and Baldwin,R. L. (1982) Annu. Rev. of a large reduction in entropy. Once Biochem. 51, 459-489 formed, however, this turn acts as a 2 Creighton,T. E. (1990) in Protein Folding nucleus to which further turns can add (Gierasch, L. M. and King, J., eds), pp. by hydrogen bonding. Thus this transform157-170, AmericanAssociationfor the ation has the property of nucleation Advancementof Science characteristic of other sharp transitions.' 3 Wetlaufer, D. B. (1990) in Protein Engineering 'Nucleus' and 'nucleation' in the model of Zimm and Bragg are structural, not kinetic. Their theory, which is very successful in treating high molecular weight homopolypeptides, deals only with the equilibria between helical and random coil forms, and not at all with the kinetics.
(Narang, S,, ed.), pp. 21-35, Butterworths 4 Somorjai, R. L. (1990) in Protein Engineering (Narang, S., ed.), pp. 1-19, Butterworths 5 Montelione,G. T. and Scheraga,H. A. (1989) Acc. Chem. Res. 22, 70-76 6 Brodsky,B. (1990)in Protein Folding (Gierasch, L. M. and King, J., eds), pp. 85-94, American Association for the Advancementof Science 7 Dyson, H. J., Lerner, R. A. and Wright, P. E. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 305-324
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
TIBS 15- NOVEMBER1990 8 Bashford, D., Karplus, M. and Weaver, D. L. (1990) in Protein Folding(Gierasch, L. M. and King, J., eds), pp. 283-290, American Association for the Advancement of Science 9 Goldberg, M. E. (1990) in Protein Folding (Gierasch, L. M. and King, J., eds), pp. 143-154, American Association for the Advancement of Science 10 Tsong, T. Y., Baldwin, R. L. and McPhie, P. (1972) ./. Mol. Biol. 63, 453-475 11 Sachs, D., Schecter, A. N., Eastlake, A. and Anfinsen, C. B. (1972) Prec. Natl Acad. Sci. USA 69, 3790-3794 22 Wetlaufer, D. B. (1973) Prec. Natl Acad. Sci. USA 70, 697-701 13 Rose, G. D., Winter, R. H. and Wetlaufer, D. B. (1976) FEBS Lett. 63, 10-16
MlCROTUBUI.I:-~; are one of the major types of filamentous protein structures of eukaryotic cells. The basic building block of microtubules is tubulin, a heterodimer made up of two related protein subunits, (z and [3 (Ref. 1). The in vitro polymerization of tubulin dimers has been well documented over the past two decades but our understanding of in vivo microtubule assembly remains very limited'. In the intracellular assembly of protein complexes, it is generally assumed that all of the information necessary for the proper folding of polypeptide chains, and their assembly into oligomeric complexes, is contained within the primary sequence of the polypeptide(s) 2. However, this dogma is now being seriously challenged by the discovery of a class of proteins referred to as 'molecular chaperones', which play an essential intermediary role in the correct folding of polypeptide chains, their intra- and extracellular transport, and their assembly into oligomeric protein complexes a,4. Two of the prominent molecular chaperone proteins that are ubiquitous belong to the chaperonin (synonyms: hsp60, GroEL homolog) and hsp70 protein families. The assembly and function of microtubules in vivo has also been tackled in my laboratory by a combined genetic and biochemical approach ~, the rationale and usefulness of which has been discussed elsewhere 7,8. These studies R. S, Gupta is at the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
14 Karplus, M. and Weaver, D. L. (1976) Nature 260, 404-406 15 Kanehisa, M. I. and Tsong, T. Y. (1978) J. Mol. Bio/. 124, 177-194 16 Wako, J. and Sato, N. (1978) J. Phys. Soc. Japan 44, 1939-1945 17 Ptitsyn, O. B. and Finkelstein, A. V. (1980) in Protein Folding (Jaenicke, R., ed.), pp. 101-106, Elsevier 18 Go, N., Abe, H., Mizuno, H. and Taketomi, H. (1980) in Protein Fo/ding(Jaenicke, R., ed.), pp. 167-181, Elsevier 19 Finkelstein, A. V. (1981) in Biomo/ecu/ar Structure, Conformation, Function, and Evolution, VoL 2 (Srinvasan, R., ed.), pp. 103-110, Pergamon Press
20 Yu, M-H. and King, J. (1984) Prec. Nat/Acad. Sci. USA 81, 6584-6588 21 Baldwin, R. L. (1986) Trends Biochem. ScL 11, 6-10 22 Baldwin, R. L. (1989) Trends Biochem. ScL 14, 291-294 23 Frank, H. S. and Wen, W-Y. (1957) Discuss. Faraday Soc. 24, 133-140 24 Gornick, F. and Hoffman, J. D. (1966) in Nucleation Phenomena, pp. 53-65, American Chemical Society 25 Flory, P. J. and Weaver, E. S. (1960) J. Am. Chem. Soc. 82, 4518-4525 26 Zimm, B. H. and Bragg, J. K. (1958) J. Chem. Phys. 28, 1246-1247 27 Wetlaufer, D. B. and Ristow, S. S. (1973) Annu. Rev. Biochem. 42, 135-158
Two proteins, P1 and P2, which are specifically altered in mammalian cell mutants resistant to antimitotic drugs, have been identified as the homologs of two members of the class of proteins known as molecular chaperones. P1 is localized in mitochondria and P2-related proteins are involved in the translocation of proteins to mitochondria. To account for these and a number of other observations, a new model for in vivo microtubule assembly is proposed. reveal that in a number of independent Chinese hamster ovary (CHO) cell mutants selected for resistance to antimitotic drugs, there is an alteration in the electrophoretic properties of two major cellular proteins, designated P1 (~60 kDa) and P2 (-70 kDa)5,6. These proteins have now been identified as homologs of chaperonin and hsp709,'°, respectively. P1 is localized within mitochondria that associate with microtubules in interphase cellsTM. These results raise a number of important questions regarding the role of mitochondria and molecular chaperone proteins in the in vivo assembly of microtubules.
Identification of P1 and P2 as homologsof the chaperonlnand hsp70 proteins Mutants expressing altered P1 and/or P2 exhibit highly specific changes in their resistance and sensitivity towards
© 1990,ElsevierSciencePublishersLtd, (UK) 0376-5067/90/$02.00
other antimitotic drugs, but no cross resistance is observed for any unrelated compounds 6,7. Reduced binding of [3H]podophyllotoxin and [SH]colchicine in cell extracts of these mutants indicate that their mutations either directly or indirectly affect binding of the antimitotic drugs to their cellular target (i.e. tubulin). Furthermore, co-release of P1, P2 and tubulin from cellular fractions under a number of different conditions (incubation at 4°C or with 5 mM CaC12) suggested that these proteins were associated with tubulin/microtubules in vivo 5,6. However, one puzzling observation that was difficult to reconcile with microtubule structure and/or assembly was that P1 and P2 were major cellular proteins, present in approximately equimolar amounts with tubulin. To investigate the cellular distribution and function of P1 and P2, specific antibodies to these proteins were
425