Centre for Protein Analysis and Design, University of Bath, Claverton Down, Bath BA2 7AY, UK
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Abstract |
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Keywords: alcohol/ß-turn/elastin/entropy/folding
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Introduction |
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Here we propose a structural model that may help to account for these observations. We have studied a temperature-induced structural transition from random coil to type II ß-turn in an elastin peptide in TFE solutions at different temperatures. The mechanism of contraction of elastin peptides derives from temperature-induced folding, also seen in the folding of cold-denaturated -helices in hexafluoro-2-propanol (HFIP) (Andersen et al., 1996
). For elastin this involves physical- (temperature/pressure) or chemical- (solvent composition) induced collapse of hydrophobically bound waters generating a positive entropy of bulk water and increased hydrophobic interactions, a phenomenon well known known in protein folding (Dill, 1990
; Livingstone et al., 1991
; Urry, 1993
; Cheng and Rossky, 1998
). Specifically, the elastin transition involves loss of hydrated water at the higher temperatures from exposed valine side chains inducing ValVal interactions that fold the VPGVG sequence into a type II ß-turn (Urry, 1988a
,b
, 1993
; Reiersen et al., 1998
). In this study, increasing temperature induced a type II ß-turn at low TFE concentrations. At higher concentrations of TFE (>30%, v/v), however, the amount of ß-turn structure decreased with increasing temperature. We propose a model in which TFE associates with elastin side chains in its cluster form and thus forms a solvent matrix which supports hydrophobic interactions.
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Results |
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Discussion |
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Some type II elastin ß-turns also fold in TFE without forming the 41 hydrogen bond (Dyson et al., 1988, and references therein; Bisaccia et al., 1998). In the sequence VPGHivG (Hiv = S-
-hydroxyvaleric acid, in which no hydrogen bond is possible), the type II CD spectrum was seen in TFE and compared well with the wild-type sequence VPGVG (Arad and Goodman, 1990
).
Rajan and Balaram (1996) have argued that TFE binds through its fluoro groups to hydrophobic side chains on -helices, although there is no experimental evidence from NMR for this (Storrs et al., 1992
). There have been observations that TFE strengthens intrahelical hydrogen bonds (Luo and Baldwin, 1997
) and weakens hydrogen bonds to solvent (Cammers-Goodwin et al., 1996
). Contrariwise, DH isotope partitioning in a GCN4 coilcoil showed that intramolecular peptide hydrogen bonds are weakened by TFE (Kentsis and Sosnick, 1998
).
The foregoing suggests that the main driving force for chemical folding of VPGVG-peptides in TFE is probably not hydrogen-bond formation. Recently, Luo and Baldwin (1999) suggested a role for hydrophobic residues in shielding polar groups from solvent in the helical state, thus stabilizing the helix against thermal denaturation (Urry, 1988a,b
, 1993
; Reiersen et al., 1998
).
Some alcohols, in particular long-chain and halogenols, form clusters that are not homogeneous in size but exist in different alcoholwater clathrate structures depending on their concentration (Kuprin et al., 1995; Hirota et al., 1998
; Gast et al., 1999
). This implies that the local concentration of TFE in these clusters may be many times larger than the bulk concentration of TFE. It is also a thermodynamic certainty that the effective TFE concentrations within the clusters will change as a function of temperature. This may affect thermodynamic calculations based on concentration and size of molecules.
TFE and HFIP have been shown to form clathrate structures starting from 10% HFIP or around 20% TFE (Kuprin et al., 1995; Gast et al., 1999
) and at even lower concentrations (Hong et al., 1999
). This implies that there are small clusters of TFE/HFIP in water with an intra-cluster alcohol concentration that is much higher than the average bulk concentration. The bell-shaped profiles of the light-scattering data show that the sizes of the clusters vary between 5 and 10 Å (Gast et al., 1999
). More recently, Hong et al. (1999) have shown by X-ray scattering that clusters of alcohols such as TFE and HFIP are formed between 0 and 80% (v/v), with a maximum scattering intensity at about 30% (v/v) for HFIP (with 14 Å clusters), although the curve for TFE is weaker and much broader. This correlated well with the transitions seen for mellitin and ß-lactoglobulin in HFIP and TFE at concentrations down to ~5% (v/v) and ~10% (v/v), respectively, for ß-lactoglobulin and even down to ~2% HFIP and ~5% TFE for mellitin. This is surprising because cluster formation at such low alcohol concentrations, frequently used for studies of protein folding, has not previously been correlated with structural transitions. However, these authors did not investigate the effect of temperature on the scattering behaviour of these cosolvents.
These data, along with our own observations, may explain the effect of TFE in supporting local hydrophobic interactions in -helices, ß-hairpins and ß-turns (Padmanabhan and Baldwin, 1994
; Albert and Hamilton, 1995
; Searle et al., 1996
; Hirota et al., 1998
; Reiersen et al., 1998
). The clusters may interact with local hydrophobic regions by disrupting water structures around these groups and provide a solvent matrix for further assisted side chain associations (Figure 3a and b
). Note that in the elastin type II ß-turn (Reiersen et al., 1998
) the valine side chains are between ~4 Å (contracted/ß-turn) and ~6 Å (extended/coil) of each other, whereas the TFE clusters are in the range 510 Å. The ability of TFE to interact with water molecules suggests that the TFE clusters can 'pull' water from the surface, at least as an initial step. This may especially be relevant at lower alcohol concentrations (010%) where the TFE clusters are not fully developed or stabilized. However, in subsequent steps the clusters may also directly associate with appropriate hydrophobic side chains. Such a matrix may be important to lower the side chain conformational entropy, which is thought to be a key factor in the folding of
-helices (Aurora et al., 1997
). The differences in cluster size and composition, dependent on TFE concentration, may explain why TFE switches secondary structure conformations at concentrations >50%. At elevated concentrations the TFE clusters are smaller, but have a higher local intra-cluster TFE concentration (Figure 3c
). This provides a better matrix to redirect non-local side chain interactions in an existing structure to a more local folding nucleus. As the majority of TFE-induced transitions occurs from a non-local (ß-sheets; Otzen and Fersth, 1995) to a local folding contact (helices, turns and hairpins; Thomas and Dill, 1993), the TFE-induced transition can easily be explained by the cluster model (Figure 3a and b
).
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The observation in this study that concentrations of TFE 30% have the opposite effect to low concentrations of TFE at higher temperatures (Figure 2a and b
versus Figure 2c and d
) suggests that the cluster structures are altered in composition and size at higher temperatures. At least for tert-butyl alcohol (Iwasaki and Fujiyama, 1979
) and hexafluoro-2-propanol (Kuprin et al., 1995
), both light scattering and low-angle X-ray scattering, respectively, have been shown to increase with temperature, possibly owing to the increased segregation of the alcohol from water (Iwasaki and Fujiyama, 1979
; Kuprin et al., 1995
). Interestingly, the scattering of clusters containing low concentrations of tert-butyl alcohol is lowered by increasing temperature (Iwasaki and Fujiyama, 1979
). At higher temperatures it would be expected that the local TFE concentration in the clusters would increase owing to a more positive mixing enthalpy between alcohol and water and that the cluster will become more hydrophobic, ultimately leading to a macroscopic phase separation (Iwasaki and Fujiyama, 1979
). This provides an explanation for the thermo-stabilization of helices whereby the TFE cluster stabilizes the helix side chains by offering a solvent matrix which can interact with the helix surface.
What can be said about cold denaturation? Cold denaturation of -helices in 810% hexafluoro-2-propanol has previously been described as a destabilization of the coil state (Andersen et al., 1996
). By reducing the temperature the density of HFIP clusters will decrease, as seen by Kuprin et al. (1995), and will therefore diminish the ability of the HFIP matrix to provide an effective local environment for the helix side chains.
The importance of having an external supporting interface in the folding and stability of proteins has been well addressed in the literature. An exposed hydrophobic surface is important for chaperone function and activity (Das and Surewicz, 1995) and
-helices are generally stabilized by tertiary hydrophobic contacts with other parts of the protein. The helix-stabilizing effect by reducing the non-polar surface area has been stressed in several previous studies (Padmanabhan and Baldwin, 1994, and references therein; Zhang et al., 1995; Butcher and Moe, 1996).
On the basis of the observations that TFE forms clusters in water solutions, we have proposed here a structural model in which TFE forms a temperature-sensitive solvent matrix that locally assists hydrophobic side chains of secondary structures. This model accounts for the various effects TFE on hydrophobic groups in peptides and proteins and may provide an explanation for the phenomenon of cold denaturation.
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Notes |
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Acknowledgments |
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References |
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---|
Andersen,N.H., Cort,J.R., Liu,Z., Sjöberg,S.J. and Tong,H. (1996) J. Am. Chem. Soc., 118, 1030910310.[ISI]
Arad,O. and Goodman,M. (1990) Biopolymers, 29, 16511668.[ISI]
Aurora,R., Creamer,T.P., Srinivasan,R. and Rose,G.D. (1997) J. Biol. Chem., 272, 14131416.
Bisaccia,F., Castiglione-Morelli,M.A., Spisani,S., Ostuni,A., Serafini-Fracassini,A., Bavoso,A. and Tamburro,A.M. (1998) Biochemistry, 37, 1112811135.[ISI][Medline]
Blanco,F.J., Jimenez,M.A., Pineda,A., Rico,M., Santoro,J. and Nieto,J.L. (1994) Biochemistry, 33, 60046014.[ISI][Medline]
Bodkin,M.J. and Goodfellow,J.M. (1996) Biopolymers, 39, 4350.[ISI][Medline]
Butcher,D.J. and Moe,G.R. (1996) Proc. Natl Acad. Sci. USA, 93, 11351140.
Cammers-Goodwin,A., Allen,T.J., Oslick,S.L., McClure,K.F., Lee,J.H. and Kemp,D.S. (1996) J. Am. Chem. Soc., 118, 30823090.[ISI]
Cheng,Y.-K. and Rossky,P.J. (1998) Nature, 392, 696699.[ISI][Medline]
Das,K.P. and Surewicz,W.K. (1995) FEBS Lett., 369, 321325.[ISI][Medline]
Dill,K.A. (1990) Biochemistry, 29, 71337155.[ISI][Medline]
Dyson,H.J., Rance,M., Houghten,R.A., Lerner,R.A. and Wright,P.E. (1988) J. Mol. Biol., 201, 161200.[ISI][Medline]
Gast,K., Zirwer,D., Müller-Frohne,M. and Damaschun,G. (1999) Protein Sci., 8, 625634.[Abstract]
Graf von Stosch,A., Jiménez,M.A., Kinzel,V. and Reed,J. (1995) Proteins: Struct. Funct. Genet., 23, 196203.[ISI][Medline]
Hirota,N., Mizuno,K. and Goto,Y. (1998) J. Mol. Biol., 275, 365378.[ISI][Medline]
Hong,D.-P., Hoshino,M., Kuboi,R. and Goto,Y. (1999) J. Am. Chem. Soc., 121, 84278433.[ISI]
Iwasaki,K. and Fujiyama, T (1979) J. Phys. Chem., 83, 463468.[ISI]
Jasanoff,A. and Fersht,A.R. (1994) Biochemistry, 33, 21292135.[ISI][Medline]
Kentsis,A. and Sosnick,T.R. (1998) Biochemistry, 37, 1461314622.[ISI][Medline]
Kuprin,S., Gräslund,A., Ehrenberg,A. and Koch,M.H.J. (1995) Biochem. Biophys. Res. Commun., 217, 11511156.[ISI][Medline]
Kuwata,K., Hoshino,M., Era,S., Batt,C.A. and Goto,Y. (1998) J. Mol. Biol., 283, 731739.[ISI][Medline]
Li,S.-C. and Deber,C.M. (1993) J. Biol. Chem., 31, 2297522978.
Livingstone,J.R., Spolar,R.S. and Record,M.T., Jr. (1991) Biochemistry, 30, 42374244.[ISI][Medline]
Lu,Z.X., Fok,K.F., Erickson,B.W. and Hugli,T.E. (1984) J. Biol. Chem., 259, 73677370.
Luo,P. and Baldwin,R.L. (1997) Biochemistry, 36, 84138421.[ISI][Medline]
Luo,P. and Baldwin,R.L. (1999) 1999) Proc. Natl Acad. Sci. USA 96, 49304935.
Luo,Y. and Baldwin,R.L. (1998) J. Mol. Biol., 279, 4957.[ISI][Medline]
Martenson,R.E., Park,J.Y. and Stone,A.L. (1985) Biochemistry, 24, 76897695.[ISI][Medline]
Narhi,L.O., Philo,J.S., Li,T., Zhang,M., Samal,B. and Arakawa,T. (1996) Biochemistry, 35, 1144711453.[ISI][Medline]
Otzen,D.E. and Fersht,A.R. (1995) Biochemistry, 34, 57185724.[ISI][Medline]
Padmanabhan,S. and Baldwin,R.L. (1994) J. Mol. Biol., 241, 706713.[ISI][Medline]
Padmanabhan,S., Jiménez,M.A., Laurents,D.V. and Rico,M. (1998) Biochemistry, 37, 1731817330.[ISI][Medline]
Perczel,A., Hollósi,M., Sándor,P. and Fasman,G.D. (1993) Int. J. Pept. Protein Res., 41, 223236.[ISI][Medline]
Rajan,R. and Balaram,P. (1996) Int. J. Pept. Protein Res., 48, 328336.[ISI][Medline]
Reiersen,H., Clarke,A.R. and Rees,A.R. (1998) J. Mol. Biol., 283, 255264.[ISI][Medline]
Searle,M.S., Zerella,R., Dudley,D.H. and Packman,L.C. (1996) Protein Eng., 9, 559565.[Abstract]
Sönnichsen,F.D., Van Eyk,J.E., Hodges,R.S. and Sykes,B.D. (1992) Biochemistry, 31, 87908798.[ISI][Medline]
Storrs,R.W., Truckses,D. and Wemmer,D.E. (1992) Biopolymers, 32, 16951702.[ISI][Medline]
Thomas,P.D. and Dill,K.A. (1993) Protein Sci., 2, 20502065.
Urry,D.W. (1988a) J. Protein Chem., 7, 134.[ISI][Medline]
Urry,D.W. (1988b) J. Protein Chem., 7, 81114.[ISI][Medline]
Urry,D.W. (1993) Angew. Chem., Int. Ed. Engl., 32, 819841.[ISI]
Walgers,R., Lee,T.C. and Cammers-Goodwin,A. (1998) J. Am. Chem. Soc., 120, 50735079.[ISI]
Wasserman,Z.R. and Salemme,F.R. (1990) Biopolymers, 29, 16131631.[ISI][Medline]
Woody,R.W. (1995) Methods Enzymol., 246, 3471.[ISI][Medline]
Yang,J.J., Pitkeathly,M. and Radford,S.E. (1994) Biochemistry, 33, 73457353.[ISI][Medline]
Zhang,H., Kaneko,K., Nguyen,J.T., Livshits,T.L., Baldwin,M.A., Cohen,F.E., James,T.L. and Prusiner,S.B. (1995) J. Mol. Biol., 250, 514526.[ISI][Medline]
Zhong,L. and Johnson,W.C., Jr. (1992) Proc. Natl Acad. Sci. USA, 89, 44624465.[Abstract]
Received July 3, 2000; revised October 11, 2000; accepted October 11, 2000.