- Email: [email protected]
Resting places on folding pathways
CHRISTOPHER 1111. DOBSON
PROTEIN
Resting places on folding
FOLDING
pathways
Recent results have reopened the controversy surrounding kinetic versus thermodynamic control of protein folding and will stimulate speculation as to the nature of barriers on folding pathways, ln recent years the mystery of how proteins fold to well defined native states has begun to be unravelled (see [ 1 ] for reviews). The notion that a protein might have to search all conformational space to be sure of ending up ti a particular state has been replaced by the idea that there are folding pathways, and indeed that there may be well-defined intermediate stages at which a protein may rest before continuing its journey. Major advances in experimental methodology have contributed to a dcveloping understanding of the structural transitions involved in folding, and these have been complemented by important progress in theoretical analysis and modelling. When a small, single domain protein is allowed to refold, it is likely that the first step is rapid collapse to a compact state, accompanied by formation of a substantial degree of secondary structure (Fig. 1). As folding proceeds, more persistent sp~itic structure, including tertiary interactions, develops until ultimately side-chains become ordered, water molecules are excluded and the native state is achieved. Native-like interactions arc believed to play a particularly important part in even the early stages of folding, although in certain cases evidence for transient non-native features has been obtained [2,3]. Foiding in vivo is likely to have many of the characteristics of in t&-o folding, though it is increasingly evident that a variety of other factors have important roles assisting protein folding in cells ([ 4j and the next article in this issue, [ 51).
~1~~1~1
Fig. 1. Possible
Volume 2
steps
native-like
in the folding
Number 7
1992
Folding of small proteins is generally fast, often being complete in seconds, and rapid reaction techniques are required to follow the processes involved. Furthermore, folding is usually highly cooperative, and well-populated kinetic intermediate states are rare. The application of methods able to provide structural details about the stages proteins go through during folding is therefore difhcult. Thus, although NMR spectroscopy is capahle of providing detailed three-dimensional structures of native proteins in solution, its application to kinetic intermediates is indirect and so far has been limited to monitoring hydrogen exchange between amides and -solvent water molecules. This last approach has, however, provided a substantial amount of information about structural changes during protein folding by detecting when different amides become protected from
or non native-like
of a hypothetical
globular
protein.
343
cofactors; and the use of very mild denaturing conditions. The most celebrated of such stable non-native states are the ‘molten globules’, commonly believed to be compact and rich in native-like secondary structure but lacking specific, well-defined tertiary interactions [8]. A key issue in the study of such states is the extent to which they can be related to genuine intermediates present on folding pathways. There is evidence that there may be similarities between molten globules and real kinetic intermediates in at least some cases [8,9], and although much remains to be learned in this area the term ‘molten globule’ is now commonly applied to kinetic as well as equilibrium states. Despite the rapidity and cooperativity of folding under most circumstances, the existence of partly folded states at equilibrium hints that it might be possible to find conditions where kinetic intermediates are trapped, even though the native state is thermodynamically stable, In this context, the recent observation [ 16; that refolding of or-lytic protease from denaturing conditions results in a non-native state that can persist for weeks at room temperature without converting to the native state is of considerable significance. Remarkably, folding to the native state from this otherwise apparently stable state takes place rapidly following addition of a 166 residue polypeptide corresponding to the amino-terminal ‘pro’ region of the inactive protease precursor. A key aspect of this study is that the incompleteiy folded state of the protein has characteristics that place it within the category of species referred to as molten globules. Other examples have been reported of proteins in states which appear to be kinetically-trapped folding intermediates. The best documented of these, however, appear to have highIy organized native-like structures, and the existence of kinetic blocks to further folding appears to be understandable in terms of the barriers to conformational change within a closely-packed structure. For example, during oxidative refolding of the protein bovine pancreatic trypsin inhibitor (BPTI), a very stable species containing only two of the three native disulphide bonds accumulates under certain circumstances. This can be attributed to the burial of the otherwise reactive cysteine thiol groups within the folded structure ([1X,12]; see [ 131 for comments on [12], and [ 141 for a response from the authors of [ 12j). Oniy when the protein is signilicantly unfolded are these thiols accessible for reaction to form the final disulphide bond; folding may therefore be enhanced by increasing the temperature or adding chemical denaturants. A further example involves refolding of serpins (protease inhibitors) which can populate a metastable active state prior to achieving the more stable latent state IlS,16]. In this case, both states of the proteins are highly folded and the cotionnational rearrangement requires a major reorganization of the P-sheet structure. In the case of the a-lytic protease intermediate, the origins of the restrictions to continued fotding are much less obvious than in the cases of the serpins and BPTI, as molten globule states are generally considered to have a high degree of conformational flexibility relative to native states. Slow interconversion within non-native states has been observed in cases where isomerization occurs about the
peptide bond preceding a proline residue [4]. Some clues that this may not be the only cause of such events have, however, started to emerge, For example, during the refolding of mutants of human lysozyme having prolines changed to other residues, slow steps in folding persist [ 171. Furthermore, the refolding of hen lysozyme has been found to occur via multiple pathways which differ in their rates of attaining the folded state [3], Again, the results are not consistent with proline isomerization being the primary cause of the heterogeneity in the folding kinetics. The case of c+tic protease di@ers, however, from other cases in the extremely slow rate of folding of the intermediate, except in the presence of the pro-region. It appears, therefore, that a-lytic protease can only continue on the folding pathway from the ‘molten globule’ state with some assistance. Has progress really stopped because the pathway has led into a dip with a high energy barrier to be surmounted before folding proceeds? Or has the path simply reached a point where the signposts have disappeared and no guide is available to prevent the selection of an incorrect route? The latter is an attractive proposition, which fits with current ideas about the role of auxiliary factors, such as molecular chaperones, which are crucial for protein folding in uiuo. Recent results suggest that such factors do indeed bind to proteins in partially folded states, such as molten globules, and facilitate folding by reducing the probability that nascent proteins follow routes that do not lead to the native state [5,18]. There is no evidence, however, that in the case of a-lytk protease the protein has any inclination to move in the wrong direction during refolding in z&w. An alternative possibility, that the effect of addition of the pro-region is to lower the height of the energy barrier to complete folding, or perhaps fmd a way around it, therefore seems more probable. How might this come about? In cases where proline isomerization hinders folding, proteins such as cyclophilin can bind and cata&= the isomerization reaction, thus speeding up folding [4]. Perhaps in a somewhat analogous way, the pro-region may bind to the partially folded state of a-lyric protease and stabilize a conformation that can convert rapidly to the native state. In doing so, as the rate of folding is increased by a factor of about 107 the pro-region would have to reduce the effective energy barrier to folding from nearly 30 to less than 10 kcal mol - 1 1lo]. The means by which this might occur are not yet understood, and for enlightenment we must await further structural information about the intermediate sute. If proteins can fall into deep wells, rather than shallow dips, during folding then the comfortable hypothesis that the native states of proteins are invariably global free energy minima must at least be open to question. The possibility that kinetics may play a serious role in determining the end product of the folding of at least .some smalI proteins under at least some circumstances will make both experimental& and theoreticians sleep a little less easily until the issue is more clearly resolved, Fortunately, the stability of the partially folded state of a-lytic protease means that the prospects for characterizing it in detail, for example by NMR spectroscopy, are good. With luck, the origin of the block to folding will then be apparent and our understanding of the structural factors that GUI @ 1992 Current Biology
cl~~yz
the details of folding pathways thereby greatly
10
BAKERD, !?OHL JL, AGARDDA: A protein folding reaction under kinetic contro1. Nature 1992, 356:263-265.
11.
STATES
. References I. CREIGHTON TE (ED): Folding and binding. Bid 1992, 2. 2,
3.
Cttrv Opin Sb-wr
SUGWAWAR TI, KuWAJMA K, SUGAI S: Folding of staphy!ococS cal nuclease A studied by equilibrium and kinetic circular dichroism spectra. RioEbemistry 1992, $0:2698-2706. REDFORD SE, D~BSON CM, EVANSPA: The folding of hen Iysowe: a complex process involving partiauy structured intermediates and multiple pathways. ~‘Vucure,in press.
4. FISCHERG, XHM~D FX: The mechanism of protein fokhng: implications of in vitro refolding models for de ltouo protein folding and translocation in the cell. Bhcbemist~ lc990, 292205-2212. 5. Emm J, CHEIGMO~’TE: Protein foIding by stagea Curr Biof 1992, 2:34?-349. 6.
UDGAONKAH
A. Nature 7.
RL NMR evidence for an early frameon the folding pathway of ribonuclease
JB, BALD~N
work intermediate 1988,
DJ, CREIGHTON TE, DOBSON CM, KARPLUS M: Conformations of intermediates in the folding of the pancreatic trypsin inhibitor. J Mel Rid 1987, 195.731-739.
12. WEISMAN JS, KIM BPTI: predominance 253:138&1393. 13.
14.
Re-examination of the folding of of native intermediates. Science 1991,
PS:
TE: Folding
CREIGHTON
256:112-l
14.
WENIAN
JS, KIM PS:
pathways of BRTL Scimcs
Reply. Science
1992,
1992,
256112-l 14.
15. CARREII.RW, EVANS DL, SIEIN PE: Mobile reactive centre of serpins and the control of thrombosis. Ntilure 1991,
353576578. 16.
~KEIGHTON
‘IX
Up the folding
pathway.
Nature
1992.
356:194-195. 17.
T. YUTANI K, T~NNAMA Y, KIRKUCHI M: Effects of proLine mutations of the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not re. sult from proline chtrans isomerization. B&&r~2fifqr 1991, 30~~2-9891.
HEWING
335:694-m.
I-L, ELVOE GA, ENG~ANDER SW: Structural characterisation of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nuture 1988, 335:700-704.
RODER
18. MARTINJ, LANGER T, B~TEVA R, SCHR~MELA, ~~ORWKH Al, HARTI FU: Chaperonin-mediated protein folding at the surhce of GroEL through a ‘molten globule’-like intermediate. Nature 1991, 352:3GJt2.
8. KUWAJ~MAK: The molten globular state as a clue for understanding the folding and cooperativity of globular protein structure. Pmtdm 1989. 687-103. 9. MIRAM
FOLDING
,
Christopher M. Dobson, Oxford Centre for Molecular Sciences, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QR, UK.
AND BINDING IN CURRENT OPINION IN STRUCTURAL
BIOLOGY
In the February 19% issue, Chris Dobson will edit the following reviews: Receptor binding by A Kosiakoff Antibody-antigen interactions by 1 Wilson Theoretical studies of proteh folding by M Karplus Physical studies of folding intermediates by R Baldwin Mutatioti studies of protein stability and folding by A Fersht Peptide conformation and folding by P Wright and J Dyson Structu~ role of accessory proteins by R Jaenicke Thermodynamics of protein stability by K Dill Denatured states of proteins by D Shortle The February
1992 issue, edited by Tom Creighton, included the following reviews:
Binding of nucleotides by proteins by GE Schulz Mutational analysis of protein stability by RT Sauer and WA Lim Unfolded proteins, compact states and molten globules by CM Dobson Metal ion binding by proteins by WA Findlay, GS Shaw and BD Sykes Role of accessory proteins in protein folding by GH Lurimer Secondary structure formation and stability by OB Ptitsyn Solvent effects on protein stability by SN Timasheff
a/oime 2 Number 7
1992
345
PROTEIN
Resting places on folding
FOLDING
pathways
Recent results have reopened the controversy surrounding kinetic versus thermodynamic control of protein folding and will stimulate speculation as to the nature of barriers on folding pathways, ln recent years the mystery of how proteins fold to well defined native states has begun to be unravelled (see [ 1 ] for reviews). The notion that a protein might have to search all conformational space to be sure of ending up ti a particular state has been replaced by the idea that there are folding pathways, and indeed that there may be well-defined intermediate stages at which a protein may rest before continuing its journey. Major advances in experimental methodology have contributed to a dcveloping understanding of the structural transitions involved in folding, and these have been complemented by important progress in theoretical analysis and modelling. When a small, single domain protein is allowed to refold, it is likely that the first step is rapid collapse to a compact state, accompanied by formation of a substantial degree of secondary structure (Fig. 1). As folding proceeds, more persistent sp~itic structure, including tertiary interactions, develops until ultimately side-chains become ordered, water molecules are excluded and the native state is achieved. Native-like interactions arc believed to play a particularly important part in even the early stages of folding, although in certain cases evidence for transient non-native features has been obtained [2,3]. Foiding in vivo is likely to have many of the characteristics of in t&-o folding, though it is increasingly evident that a variety of other factors have important roles assisting protein folding in cells ([ 4j and the next article in this issue, [ 51).
~1~~1~1
Fig. 1. Possible
Volume 2
steps
native-like
in the folding
Number 7
1992
Folding of small proteins is generally fast, often being complete in seconds, and rapid reaction techniques are required to follow the processes involved. Furthermore, folding is usually highly cooperative, and well-populated kinetic intermediate states are rare. The application of methods able to provide structural details about the stages proteins go through during folding is therefore difhcult. Thus, although NMR spectroscopy is capahle of providing detailed three-dimensional structures of native proteins in solution, its application to kinetic intermediates is indirect and so far has been limited to monitoring hydrogen exchange between amides and -solvent water molecules. This last approach has, however, provided a substantial amount of information about structural changes during protein folding by detecting when different amides become protected from
or non native-like
of a hypothetical
globular
protein.
343
cofactors; and the use of very mild denaturing conditions. The most celebrated of such stable non-native states are the ‘molten globules’, commonly believed to be compact and rich in native-like secondary structure but lacking specific, well-defined tertiary interactions [8]. A key issue in the study of such states is the extent to which they can be related to genuine intermediates present on folding pathways. There is evidence that there may be similarities between molten globules and real kinetic intermediates in at least some cases [8,9], and although much remains to be learned in this area the term ‘molten globule’ is now commonly applied to kinetic as well as equilibrium states. Despite the rapidity and cooperativity of folding under most circumstances, the existence of partly folded states at equilibrium hints that it might be possible to find conditions where kinetic intermediates are trapped, even though the native state is thermodynamically stable, In this context, the recent observation [ 16; that refolding of or-lytic protease from denaturing conditions results in a non-native state that can persist for weeks at room temperature without converting to the native state is of considerable significance. Remarkably, folding to the native state from this otherwise apparently stable state takes place rapidly following addition of a 166 residue polypeptide corresponding to the amino-terminal ‘pro’ region of the inactive protease precursor. A key aspect of this study is that the incompleteiy folded state of the protein has characteristics that place it within the category of species referred to as molten globules. Other examples have been reported of proteins in states which appear to be kinetically-trapped folding intermediates. The best documented of these, however, appear to have highIy organized native-like structures, and the existence of kinetic blocks to further folding appears to be understandable in terms of the barriers to conformational change within a closely-packed structure. For example, during oxidative refolding of the protein bovine pancreatic trypsin inhibitor (BPTI), a very stable species containing only two of the three native disulphide bonds accumulates under certain circumstances. This can be attributed to the burial of the otherwise reactive cysteine thiol groups within the folded structure ([1X,12]; see [ 131 for comments on [12], and [ 141 for a response from the authors of [ 12j). Oniy when the protein is signilicantly unfolded are these thiols accessible for reaction to form the final disulphide bond; folding may therefore be enhanced by increasing the temperature or adding chemical denaturants. A further example involves refolding of serpins (protease inhibitors) which can populate a metastable active state prior to achieving the more stable latent state IlS,16]. In this case, both states of the proteins are highly folded and the cotionnational rearrangement requires a major reorganization of the P-sheet structure. In the case of the a-lytic protease intermediate, the origins of the restrictions to continued fotding are much less obvious than in the cases of the serpins and BPTI, as molten globule states are generally considered to have a high degree of conformational flexibility relative to native states. Slow interconversion within non-native states has been observed in cases where isomerization occurs about the
peptide bond preceding a proline residue [4]. Some clues that this may not be the only cause of such events have, however, started to emerge, For example, during the refolding of mutants of human lysozyme having prolines changed to other residues, slow steps in folding persist [ 171. Furthermore, the refolding of hen lysozyme has been found to occur via multiple pathways which differ in their rates of attaining the folded state [3], Again, the results are not consistent with proline isomerization being the primary cause of the heterogeneity in the folding kinetics. The case of c+tic protease di@ers, however, from other cases in the extremely slow rate of folding of the intermediate, except in the presence of the pro-region. It appears, therefore, that a-lytic protease can only continue on the folding pathway from the ‘molten globule’ state with some assistance. Has progress really stopped because the pathway has led into a dip with a high energy barrier to be surmounted before folding proceeds? Or has the path simply reached a point where the signposts have disappeared and no guide is available to prevent the selection of an incorrect route? The latter is an attractive proposition, which fits with current ideas about the role of auxiliary factors, such as molecular chaperones, which are crucial for protein folding in uiuo. Recent results suggest that such factors do indeed bind to proteins in partially folded states, such as molten globules, and facilitate folding by reducing the probability that nascent proteins follow routes that do not lead to the native state [5,18]. There is no evidence, however, that in the case of a-lytk protease the protein has any inclination to move in the wrong direction during refolding in z&w. An alternative possibility, that the effect of addition of the pro-region is to lower the height of the energy barrier to complete folding, or perhaps fmd a way around it, therefore seems more probable. How might this come about? In cases where proline isomerization hinders folding, proteins such as cyclophilin can bind and cata&= the isomerization reaction, thus speeding up folding [4]. Perhaps in a somewhat analogous way, the pro-region may bind to the partially folded state of a-lyric protease and stabilize a conformation that can convert rapidly to the native state. In doing so, as the rate of folding is increased by a factor of about 107 the pro-region would have to reduce the effective energy barrier to folding from nearly 30 to less than 10 kcal mol - 1 1lo]. The means by which this might occur are not yet understood, and for enlightenment we must await further structural information about the intermediate sute. If proteins can fall into deep wells, rather than shallow dips, during folding then the comfortable hypothesis that the native states of proteins are invariably global free energy minima must at least be open to question. The possibility that kinetics may play a serious role in determining the end product of the folding of at least .some smalI proteins under at least some circumstances will make both experimental& and theoreticians sleep a little less easily until the issue is more clearly resolved, Fortunately, the stability of the partially folded state of a-lytic protease means that the prospects for characterizing it in detail, for example by NMR spectroscopy, are good. With luck, the origin of the block to folding will then be apparent and our understanding of the structural factors that GUI @ 1992 Current Biology
cl~~yz
the details of folding pathways thereby greatly
10
BAKERD, !?OHL JL, AGARDDA: A protein folding reaction under kinetic contro1. Nature 1992, 356:263-265.
11.
STATES
. References I. CREIGHTON TE (ED): Folding and binding. Bid 1992, 2. 2,
3.
Cttrv Opin Sb-wr
SUGWAWAR TI, KuWAJMA K, SUGAI S: Folding of staphy!ococS cal nuclease A studied by equilibrium and kinetic circular dichroism spectra. RioEbemistry 1992, $0:2698-2706. REDFORD SE, D~BSON CM, EVANSPA: The folding of hen Iysowe: a complex process involving partiauy structured intermediates and multiple pathways. ~‘Vucure,in press.
4. FISCHERG, XHM~D FX: The mechanism of protein fokhng: implications of in vitro refolding models for de ltouo protein folding and translocation in the cell. Bhcbemist~ lc990, 292205-2212. 5. Emm J, CHEIGMO~’TE: Protein foIding by stagea Curr Biof 1992, 2:34?-349. 6.
UDGAONKAH
A. Nature 7.
RL NMR evidence for an early frameon the folding pathway of ribonuclease
JB, BALD~N
work intermediate 1988,
DJ, CREIGHTON TE, DOBSON CM, KARPLUS M: Conformations of intermediates in the folding of the pancreatic trypsin inhibitor. J Mel Rid 1987, 195.731-739.
12. WEISMAN JS, KIM BPTI: predominance 253:138&1393. 13.
14.
Re-examination of the folding of of native intermediates. Science 1991,
PS:
TE: Folding
CREIGHTON
256:112-l
14.
WENIAN
JS, KIM PS:
pathways of BRTL Scimcs
Reply. Science
1992,
1992,
256112-l 14.
15. CARREII.RW, EVANS DL, SIEIN PE: Mobile reactive centre of serpins and the control of thrombosis. Ntilure 1991,
353576578. 16.
~KEIGHTON
‘IX
Up the folding
pathway.
Nature
1992.
356:194-195. 17.
T. YUTANI K, T~NNAMA Y, KIRKUCHI M: Effects of proLine mutations of the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not re. sult from proline chtrans isomerization. B&&r~2fifqr 1991, 30~~2-9891.
HEWING
335:694-m.
I-L, ELVOE GA, ENG~ANDER SW: Structural characterisation of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nuture 1988, 335:700-704.
RODER
18. MARTINJ, LANGER T, B~TEVA R, SCHR~MELA, ~~ORWKH Al, HARTI FU: Chaperonin-mediated protein folding at the surhce of GroEL through a ‘molten globule’-like intermediate. Nature 1991, 352:3GJt2.
8. KUWAJ~MAK: The molten globular state as a clue for understanding the folding and cooperativity of globular protein structure. Pmtdm 1989. 687-103. 9. MIRAM
FOLDING
,
Christopher M. Dobson, Oxford Centre for Molecular Sciences, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QR, UK.
AND BINDING IN CURRENT OPINION IN STRUCTURAL
BIOLOGY
In the February 19% issue, Chris Dobson will edit the following reviews: Receptor binding by A Kosiakoff Antibody-antigen interactions by 1 Wilson Theoretical studies of proteh folding by M Karplus Physical studies of folding intermediates by R Baldwin Mutatioti studies of protein stability and folding by A Fersht Peptide conformation and folding by P Wright and J Dyson Structu~ role of accessory proteins by R Jaenicke Thermodynamics of protein stability by K Dill Denatured states of proteins by D Shortle The February
1992 issue, edited by Tom Creighton, included the following reviews:
Binding of nucleotides by proteins by GE Schulz Mutational analysis of protein stability by RT Sauer and WA Lim Unfolded proteins, compact states and molten globules by CM Dobson Metal ion binding by proteins by WA Findlay, GS Shaw and BD Sykes Role of accessory proteins in protein folding by GH Lurimer Secondary structure formation and stability by OB Ptitsyn Solvent effects on protein stability by SN Timasheff
a/oime 2 Number 7
1992
345