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Structure and stability of proteins: The role of solvent
10 (1984) Publishers B.V.,
Colloids and Surfaces, Elsevier
Science
STRUCTURE
JOEL
l-7 Amsterdam
AND STABILITY
-Printed
in The Netherlands
OF PROTEINS:
THE ROLE
OF SOLVENT
JANIN
Laboratoire de Biologie Physicochimique, Orsay (France) (Received
27 October
1983;
accepted
Ba^timent 433, Uniuersite’ Paris-Sud,
22 December
91405
1983)
ABSTRACT Solvent is involved in the stability of proteins through protein-solvent interactions and through the hydrophobic effect. Water molecules bound to polar groups on proteins can be observed in X-ray crystal structures, while the role of hydrophobicity is inferred from thermodynamic studies and computer simulations. In addition to water, lipid bilayers are a natural solvent for membrane proteins. Their properties are compared to those of water-soluble proteins.
INTRODUCTION
Proteins are unusual among organic polymers in having a single conformation, or a small number of closely related ones which are commonly called the native state. The native state is responsible for the biological activity, and its stability is strongly dependent on the environment, including the presence of water as a solvent for most proteins, or of lipids for membrane proteins. Interactions between protein and solvent and, indirectly, interactions between solvent molecules, contribute to the stability of the native state. They have been the object of structural, physical and chemical studies, and, recently, they have been studied using computer simulation by molecular mechanics or dynamics studies. BACKGROUND
Native us denatured
state
The protein is in equilibrium between its uniquely defined native state and the many conformations of the unfolded polypeptide chain which constitute the denatured state. The transition from native to denatured is easily observed by raising the temperature, and the corresponding AGO and AH0 can be measured. The measurement is meaningful only if the transition is reversible. In most cases, this requires an environment where the unfolded chains do not precipitate, as in high concentrations of urea or guanidinium
0166-6622/84/$03.00
o 1984
Elsevier
Science
Publishers
B.V.
2
chloride. Following Tanford [ 11, many authors have studied the reversible transition from the native state in water to the denatured state in various concentrations of denaturants [ 21. Thermodynamic measurements are then extrapolated to zero concentration of denaturant. Though each protein behaves differently in such studies, the following general rules apply: (a) The native state is only marginally stable. Its Gibbs energy is only 515 kcal mol-’ less than for the denatured state. (b) Its stability results from a compromise between large entropic and enthalpic effects. Thus, denaturation AH’s, measured using the van’t Hoff law or by calorimetry [ 3, 41 are often larger than 100 kcal mol-‘. (c) The transition is highly cooperative. This is because to a good approximation, only two states are observed for small proteins. In proteins with several hundred amino acid residues, regions of the polypeptide chain that fold cooperatively can often be defined (domains) [ 5, 61. The results are compatible with a thermodynamic description of protein folding where the large conformational entropy of the unfolded chain favours the denatured state, while intramolecular interactions favour the native folded state. The strength of these interactions in vacua has been estimated from molecular mechanics [ 7, 81, which leads to values of some 10 kcal mol-’ per residue for stabilizing van der Waals and hydrogen bond enthalpies in native globular proteins. However, these values are obtained in energyminimized conformations that deviate somewhat from actual X-ray structures. They should be compared with the corresponding enthalpy of proteinsolvent interactions, of which there are many more in unfolded conformations. Intramolecular interactions are certainly not a sufficient explanation for the stability of proteins, and we shall see that solvent entropy plays a major role through the hydrophobic effect. RESULTS
Polar interactions
between protein
and solvent
Protein-solvent interactions are easier to assess in the native state, where the protein is stable while the solvent fluctuates. High resolution X-ray data are an important source of information on bound water molecules at the protein surface and, exceptionally, within the protein. The mean position of the oxygen atom and (through the Debye-Waller temperature factor) its mean displacement can be measured, provided the latter is less than about 1 i$ . When neutron diffraction data are available, the orientation of the H,O molecules can also be determined [ 91. Water molecules bound to part of crystalline insulin are shown in Fig. 1. Some of them are seen to form H-bonds to protein polar groups, and H-bond in turn to other water molecules. Quantitative descriptions of solvent structure in a fungal pepsin [lo], and in two crystalline lysozymes [ 111, have recently been published. Of the many water molecules (several hundreds) that can be localized with
3
Fig. 1. Water molecules bound to crystalline insulin. Part of the 1.5 A-resolution electron density map of rhombohedral porcine insulin, with the protein main chain (dark bonds) and surrounding water molecules attached to it through possible H-bonds (dashes). Two main-chain )C=O groups and two )NH groups are seen to be involved. Other water molecules are immobilized through a network of H-bonds involving the first layer of water bound to the protein. In crystalline insulin, most of the water contained in the crystal cell is caught in this network of H-bonds and can be localized in the electron density map. Taken from Y. Mauguen, Ph.D. Thesis, Universite Paris-Sud (1979).
certainty in their electron density maps, 80% are directly bound to protein polar groups, with a strong preference for oxygen over nitrogen as H-bond donors or acceptors. Main-chain peptide X=0 and )NH groups are more often (60%) involved than side-chain polar groups; yet most if not all polar groups placed on the protein surface form H-bonds to the solvent. The geometry of these bonds is close to that of model compounds, but so is the geometry of the water-water bonds made by the other 20% of water molecules that are not directly bonded to the protein, yet have stable positions in the crystal structures. The finding that the Debye-Waller temperature factors of immobilized water molecules are about the same as those of neighbouring protein atoms [ll] also argues for a stable association of water to protein
4
polar groups. In contrast, no (positional) water ordering is detected near protein non-polar groups, and no clathrate-like structures, though clathrates have sometimes been proposed as models for protein-water association. Isotopic exchange studies indicate that bound water molecules are in rapid exchange with bulk water, though they are no longer in a liquid state. They constitute the “unfreezable water” fraction that cannot be lyophilized away from proteins, or the “bound solvent” observed in hydrodynamic studies, which often amounts to 30-60s of the protein mass [ 121, Knowing the three-dimensional structure, the total amount of water molecules bound in the first layer can be estimated from the area of the protein polar surface that is accessible to the solvent. This area was calculated by a geometric procedure due to Lee and Richards [13,14]. For a small protein like the pancreatic trypsin inhibitor, it is 820 .A*, enough for 82 water molecules to bind, or about 25% of the protein mass [15]. The number of water molecules that have been localized in the crystal structure of the inhibitor [ 161, is of the same order. In crystalline human lysozyme [ 111, 143 water molecules are identified. They cover 75% of the available protein surface. Given that a fraction of the surface is involved in crystal contacts between protein molecules, the total number of bound water molecules in solution should be about 230, or 35% of the protein mass. Estimates of lysozyme hydration in solution agree with this number. Solvent-protein H-bonds can have a stabilizing effect on the protein provided the binding enthalpy is significantly larger than the free.energy lost by immobilized solvent molecules. Molecular dynamics [ 81 and Monte-Carlo simulations [ 171 performed on water indicate that the presence of protein generates potential energy wells which may be 5-10 kcal mol-’ deep, when more than one H-bond is formed. It is, therefore, reasonable to assume that a water molecule that bridges the protein surface by making more than one H-bond, contributes to its stability. Interestingly, water molecules are found to bridge subunits in haemoglobin [ 181, where they occur at the allosteric interface, and may stabilize the deoxy form of tetrameric haemoglobin. The hydrophobic
effect
Though there is no evidence for clathrate-like positional ordering of solvent around non-polar groups on the protein surface, no H-bond can be made by water molecules pointing towards this surface and their orientation is restricted, hence, their entropy increases. Computer simulations confirm this property of non-polar contacts, which is assumed here to be the physical basis for the so-called hydrophobic effect. Quantitative estimates of the effect are obtained on small molecules by measuring their free energy of transfer between aqueous and non-polar solvents or to the gas phase. Such measurements are not feasible on macromolecules, for which we rely on the empirical observation made by Hermann [ 191, and Chothia [ 201: free energies of transfer vary linearly with the area of the molecular
5
surface accessible to the solvent (or with the area of the water-molecule contact surface, which is closely related to it [ 141). This relationship should apply to non-polar surfaces according to our interpretation of the hydrophobic effect. The molecular surface of native soluble proteins is largely non-polar (5060%). It is actually no more polar than the unfolded polypeptide chain in spite of the well-known tendency for non-polar amino-acid residues to pack inside the three-dimensional structure. This paradox disappears when we consider the hierarchy of levels in protein structure [ 211. The polypeptide main chain is largely polar like the peptide group itself, while most aminoacid side chains are non-polar. Formation of secondary structure (a-helices and P-sheets) buries a large amount of main chain, which forms peptide-topeptide H-bonds. The surface of ol-helices and o-sheets is less polar than that of the unfolded chain, with non-polar groups forming up to 70% of their accessible surface [ 151. Association of these structural elements into a globular tertiary structure buries a large fraction of the non-polar surface into a-helix/a-helix contacts, a-helix/p-sheets contacts, or p-sheet/P-sheet contacts. The geometry of each sort of contact is characteristic of a given class of tertiary structures [22] . However, they have in common that contacts between these structural elements exclude H-bonds, except at the periphery, and bury a large amount of non-polar surface [ 151. This suggests that the hydrophobic effect is the major driving factor in associating cu-helices and/or p-sheets, and that, while secondary structure is stabilized by H-bond (though not only by H-bonds), tertiary structure is stabilized by the hydrophobic effect. Calibrations indicate that the corresponding A GG is about 25 cal mall’ per A2 of protein-accessible surface area [ 231, which leads to AGO = 5-15 kcal mol-’ for surface areas of 200-600 8’ that are removed from contact with the solvent when two structural elements associate. This contributes in favour of the folded state to the Gibbs energy of protein denaturation.
Proteins in a non-aqueous
environment
Small monomeric proteins are globular in the sense that they achieve a low surface-to-volume ratio. Thus, even though more than half of the residual surface in contact with the solvent may be non-polar, folding reduces it to a minimum [ 221. This rule does not apply to polypeptide chains of molecular weight >30 000, which fold in domains. They have non-polar regions involved in intermolecular contacts at their surface, leading to polymerization, either into oligomers (dimers, tetramers, etc.), or into structures with helical repeats (muscles fibres, microtubules, virus). Inter-subunit contacts in protein oligomers have been analyzed. We find both non-polar and polar surfaces to be involved, H-bonding being an obligatory feature of the second [ 241. The same rules apply to the formation of helical and spherical verus capsids. In fibers of sickle-cell haemoglobin, non-polar contacts are dominant.
6
In large aggregates such as fibres, most of the protein surface is no longer in an aqueous environment. This is even more true of a membrane protein, part of which is in a lipid environment whose physicochemical properties are basically different from those of water. A lipid bilayer is ~60 .4 thick, and is non-polar over little more than 30 8, corresponding to the fatty acid chains. Thus, membrane proteins are embedded in a 30 .&-deep non-polar lipid “solvent”. The rest of their surface is in contact with the polar head groups of the lipids or with water on either side of the bilayer. For an integral membrane protein such as bacteriorhodopsin [ 251, which crosses the bilayer, about half of a total accessible surface of some 9000 A2 is estimated to be in a non-polar environment (exact numbers will be available when the detailed three-dimensional structure is known from electron diffraction studies).’ This fraction of the protein surface must be non-polar. Assuming that the other half, in contact with lipid head groups or with water, has the same average properties as for soluble proteins, then 70430% of the bacteriorhodopsin surface rather than the usual 50-60% must be non-polar. This is reflected in its amino-acid composition, typical of integral membrane proteins, with long stretches of non-polar residues. Bacteriorhodopsin folds into ol-helices which pack in the usual ways [ 261, and the “inside” of this protein is not expected to be more polar than that of globular proteins, except where required by its function as a proton channel. Thus, the author does not consider the ‘“inverted protein” structure proposed for membrane proteins to be significant:A specific feature of membrane proteins is the non-polar ring on their surface, not their polar inside. In bacteriorhodopsin, the ring circles the whole protein. In others, it may form the surface of a single a-helix, or of a pair of cu-helices if we believe models [27, 281, which have been proposed for the folding or hydrophobic “signal peptides” that help excreted proteins to pass through membranes while they are synthesized
[=I. CONCLUSIONS
Water is the native environment of most proteins. It affects their structure through the direct and indirect effects analyzed here. It is also involved in function, for instance as a substrate in many enzymatic reactions. The same factors, polar interactions and the hydrophobic effect, which are important in stabilizing proteins, are also active iii promoting ligand binding to active sites, and in building elaborate multi-molecular structures which, like muscle fibres, may reach macroscopic dimensions.
REFERENCES 1 2
C. Tanford, Adv. Protein Chem., 23 (1968) 121. T.E. Creighton, Prog. Biophys. Mol. Biol., 33 (1979)
231-297.
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
P.L. Privalov, Prog. Biophys. Mol. Biol., 33 (1979) 167-241. P.L. Privalov, Prog. Biophys. Mol. Biol., 35 (1982) l-104. D.B. Wetlaufer, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 696-701. J. Janin and S. Wodak, Prog. Biophys. Mol. Biol., 42 (1983) 21-78. M. Levitt, in R. Jaenicke (Ed.), Protein Folding, Elsevier, Amsterdam, 1980, pp. 1736. J.A. M&ammon and M. Karplus, CRC Crit. Rev. Biochem., 9 (1981) 293-349. A. Wlodawer, Prog. Biophys. Mol. Biol., 40 (1982) 115-159. M.N.G. James and A.R. Sielecki, J. Mol. Biol., 163 (1983) 299-361. C.C.F. Blake, W.C.A. Pulford and P.J. Artymiuck, J. Mol. Biol., 167 (1983) 693723. I.D. Kuntz and W. Kaufmann, Adv. Protein Chem., 28 (1974) 239-345. B.K. Lee and F.M. Richards, J. Mol. Biol., 55 (1971) 379-400. F.M. Richards, Ann. Rev. Biophys. Bioeng., 6 (1977) 151-176. C. Chothia, J. Mol. Biol., 105 (1976) l-14. J. Deisenhofer and W. Steigemann, Acta Crystallogr. Sect. B, 31 (1975) 238-250. A.T. Hagler and J. Moult, Nature (London), 272 (1978) 222-226. G. Fermi, J. Mol. Biol., 97 (1975) 237-256. R.B. Herrmann, J. Phys. Chem., 76 (1972) 2754-2759. C. Chothia, Nature (London), 248 (1974) 338-339. G.E. Schultz and R.H. Schirmer, Principles of Protein Structure, Springer, Heidelberg, 1979. J. Janin, Bull. Inst. Pasteur, 77 (1979) 337-373. C. Chothia, Nature (London), 254 (1975) 304-308. C. Chothia and J. Janin, Nature (London), 256 (1976) 705-708. W. Stoeckenius, R.H. Lozier and R.A. Bogomolni, Biochim. Biophys. Acta, 505 (1979) 215-278. R. Henderson and P.N.T. Unwin, Nature (London), 257 (1975) 28-32. J. Garnier, P. Gaye, J.C. Mercier and B. Robson, Biochimie, 62 (1980) 231-239. D.M. Engelman and T.A. Steitz, Cell, 23 (1981) 411-422. G. Blobell and D. Dobberstein, J. Cell. Biol., 67 (1975) 835-851.
Colloids and Surfaces, Elsevier
Science
STRUCTURE
JOEL
l-7 Amsterdam
AND STABILITY
-Printed
in The Netherlands
OF PROTEINS:
THE ROLE
OF SOLVENT
JANIN
Laboratoire de Biologie Physicochimique, Orsay (France) (Received
27 October
1983;
accepted
Ba^timent 433, Uniuersite’ Paris-Sud,
22 December
91405
1983)
ABSTRACT Solvent is involved in the stability of proteins through protein-solvent interactions and through the hydrophobic effect. Water molecules bound to polar groups on proteins can be observed in X-ray crystal structures, while the role of hydrophobicity is inferred from thermodynamic studies and computer simulations. In addition to water, lipid bilayers are a natural solvent for membrane proteins. Their properties are compared to those of water-soluble proteins.
INTRODUCTION
Proteins are unusual among organic polymers in having a single conformation, or a small number of closely related ones which are commonly called the native state. The native state is responsible for the biological activity, and its stability is strongly dependent on the environment, including the presence of water as a solvent for most proteins, or of lipids for membrane proteins. Interactions between protein and solvent and, indirectly, interactions between solvent molecules, contribute to the stability of the native state. They have been the object of structural, physical and chemical studies, and, recently, they have been studied using computer simulation by molecular mechanics or dynamics studies. BACKGROUND
Native us denatured
state
The protein is in equilibrium between its uniquely defined native state and the many conformations of the unfolded polypeptide chain which constitute the denatured state. The transition from native to denatured is easily observed by raising the temperature, and the corresponding AGO and AH0 can be measured. The measurement is meaningful only if the transition is reversible. In most cases, this requires an environment where the unfolded chains do not precipitate, as in high concentrations of urea or guanidinium
0166-6622/84/$03.00
o 1984
Elsevier
Science
Publishers
B.V.
2
chloride. Following Tanford [ 11, many authors have studied the reversible transition from the native state in water to the denatured state in various concentrations of denaturants [ 21. Thermodynamic measurements are then extrapolated to zero concentration of denaturant. Though each protein behaves differently in such studies, the following general rules apply: (a) The native state is only marginally stable. Its Gibbs energy is only 515 kcal mol-’ less than for the denatured state. (b) Its stability results from a compromise between large entropic and enthalpic effects. Thus, denaturation AH’s, measured using the van’t Hoff law or by calorimetry [ 3, 41 are often larger than 100 kcal mol-‘. (c) The transition is highly cooperative. This is because to a good approximation, only two states are observed for small proteins. In proteins with several hundred amino acid residues, regions of the polypeptide chain that fold cooperatively can often be defined (domains) [ 5, 61. The results are compatible with a thermodynamic description of protein folding where the large conformational entropy of the unfolded chain favours the denatured state, while intramolecular interactions favour the native folded state. The strength of these interactions in vacua has been estimated from molecular mechanics [ 7, 81, which leads to values of some 10 kcal mol-’ per residue for stabilizing van der Waals and hydrogen bond enthalpies in native globular proteins. However, these values are obtained in energyminimized conformations that deviate somewhat from actual X-ray structures. They should be compared with the corresponding enthalpy of proteinsolvent interactions, of which there are many more in unfolded conformations. Intramolecular interactions are certainly not a sufficient explanation for the stability of proteins, and we shall see that solvent entropy plays a major role through the hydrophobic effect. RESULTS
Polar interactions
between protein
and solvent
Protein-solvent interactions are easier to assess in the native state, where the protein is stable while the solvent fluctuates. High resolution X-ray data are an important source of information on bound water molecules at the protein surface and, exceptionally, within the protein. The mean position of the oxygen atom and (through the Debye-Waller temperature factor) its mean displacement can be measured, provided the latter is less than about 1 i$ . When neutron diffraction data are available, the orientation of the H,O molecules can also be determined [ 91. Water molecules bound to part of crystalline insulin are shown in Fig. 1. Some of them are seen to form H-bonds to protein polar groups, and H-bond in turn to other water molecules. Quantitative descriptions of solvent structure in a fungal pepsin [lo], and in two crystalline lysozymes [ 111, have recently been published. Of the many water molecules (several hundreds) that can be localized with
3
Fig. 1. Water molecules bound to crystalline insulin. Part of the 1.5 A-resolution electron density map of rhombohedral porcine insulin, with the protein main chain (dark bonds) and surrounding water molecules attached to it through possible H-bonds (dashes). Two main-chain )C=O groups and two )NH groups are seen to be involved. Other water molecules are immobilized through a network of H-bonds involving the first layer of water bound to the protein. In crystalline insulin, most of the water contained in the crystal cell is caught in this network of H-bonds and can be localized in the electron density map. Taken from Y. Mauguen, Ph.D. Thesis, Universite Paris-Sud (1979).
certainty in their electron density maps, 80% are directly bound to protein polar groups, with a strong preference for oxygen over nitrogen as H-bond donors or acceptors. Main-chain peptide X=0 and )NH groups are more often (60%) involved than side-chain polar groups; yet most if not all polar groups placed on the protein surface form H-bonds to the solvent. The geometry of these bonds is close to that of model compounds, but so is the geometry of the water-water bonds made by the other 20% of water molecules that are not directly bonded to the protein, yet have stable positions in the crystal structures. The finding that the Debye-Waller temperature factors of immobilized water molecules are about the same as those of neighbouring protein atoms [ll] also argues for a stable association of water to protein
4
polar groups. In contrast, no (positional) water ordering is detected near protein non-polar groups, and no clathrate-like structures, though clathrates have sometimes been proposed as models for protein-water association. Isotopic exchange studies indicate that bound water molecules are in rapid exchange with bulk water, though they are no longer in a liquid state. They constitute the “unfreezable water” fraction that cannot be lyophilized away from proteins, or the “bound solvent” observed in hydrodynamic studies, which often amounts to 30-60s of the protein mass [ 121, Knowing the three-dimensional structure, the total amount of water molecules bound in the first layer can be estimated from the area of the protein polar surface that is accessible to the solvent. This area was calculated by a geometric procedure due to Lee and Richards [13,14]. For a small protein like the pancreatic trypsin inhibitor, it is 820 .A*, enough for 82 water molecules to bind, or about 25% of the protein mass [15]. The number of water molecules that have been localized in the crystal structure of the inhibitor [ 161, is of the same order. In crystalline human lysozyme [ 111, 143 water molecules are identified. They cover 75% of the available protein surface. Given that a fraction of the surface is involved in crystal contacts between protein molecules, the total number of bound water molecules in solution should be about 230, or 35% of the protein mass. Estimates of lysozyme hydration in solution agree with this number. Solvent-protein H-bonds can have a stabilizing effect on the protein provided the binding enthalpy is significantly larger than the free.energy lost by immobilized solvent molecules. Molecular dynamics [ 81 and Monte-Carlo simulations [ 171 performed on water indicate that the presence of protein generates potential energy wells which may be 5-10 kcal mol-’ deep, when more than one H-bond is formed. It is, therefore, reasonable to assume that a water molecule that bridges the protein surface by making more than one H-bond, contributes to its stability. Interestingly, water molecules are found to bridge subunits in haemoglobin [ 181, where they occur at the allosteric interface, and may stabilize the deoxy form of tetrameric haemoglobin. The hydrophobic
effect
Though there is no evidence for clathrate-like positional ordering of solvent around non-polar groups on the protein surface, no H-bond can be made by water molecules pointing towards this surface and their orientation is restricted, hence, their entropy increases. Computer simulations confirm this property of non-polar contacts, which is assumed here to be the physical basis for the so-called hydrophobic effect. Quantitative estimates of the effect are obtained on small molecules by measuring their free energy of transfer between aqueous and non-polar solvents or to the gas phase. Such measurements are not feasible on macromolecules, for which we rely on the empirical observation made by Hermann [ 191, and Chothia [ 201: free energies of transfer vary linearly with the area of the molecular
5
surface accessible to the solvent (or with the area of the water-molecule contact surface, which is closely related to it [ 141). This relationship should apply to non-polar surfaces according to our interpretation of the hydrophobic effect. The molecular surface of native soluble proteins is largely non-polar (5060%). It is actually no more polar than the unfolded polypeptide chain in spite of the well-known tendency for non-polar amino-acid residues to pack inside the three-dimensional structure. This paradox disappears when we consider the hierarchy of levels in protein structure [ 211. The polypeptide main chain is largely polar like the peptide group itself, while most aminoacid side chains are non-polar. Formation of secondary structure (a-helices and P-sheets) buries a large amount of main chain, which forms peptide-topeptide H-bonds. The surface of ol-helices and o-sheets is less polar than that of the unfolded chain, with non-polar groups forming up to 70% of their accessible surface [ 151. Association of these structural elements into a globular tertiary structure buries a large fraction of the non-polar surface into a-helix/a-helix contacts, a-helix/p-sheets contacts, or p-sheet/P-sheet contacts. The geometry of each sort of contact is characteristic of a given class of tertiary structures [22] . However, they have in common that contacts between these structural elements exclude H-bonds, except at the periphery, and bury a large amount of non-polar surface [ 151. This suggests that the hydrophobic effect is the major driving factor in associating cu-helices and/or p-sheets, and that, while secondary structure is stabilized by H-bond (though not only by H-bonds), tertiary structure is stabilized by the hydrophobic effect. Calibrations indicate that the corresponding A GG is about 25 cal mall’ per A2 of protein-accessible surface area [ 231, which leads to AGO = 5-15 kcal mol-’ for surface areas of 200-600 8’ that are removed from contact with the solvent when two structural elements associate. This contributes in favour of the folded state to the Gibbs energy of protein denaturation.
Proteins in a non-aqueous
environment
Small monomeric proteins are globular in the sense that they achieve a low surface-to-volume ratio. Thus, even though more than half of the residual surface in contact with the solvent may be non-polar, folding reduces it to a minimum [ 221. This rule does not apply to polypeptide chains of molecular weight >30 000, which fold in domains. They have non-polar regions involved in intermolecular contacts at their surface, leading to polymerization, either into oligomers (dimers, tetramers, etc.), or into structures with helical repeats (muscles fibres, microtubules, virus). Inter-subunit contacts in protein oligomers have been analyzed. We find both non-polar and polar surfaces to be involved, H-bonding being an obligatory feature of the second [ 241. The same rules apply to the formation of helical and spherical verus capsids. In fibers of sickle-cell haemoglobin, non-polar contacts are dominant.
6
In large aggregates such as fibres, most of the protein surface is no longer in an aqueous environment. This is even more true of a membrane protein, part of which is in a lipid environment whose physicochemical properties are basically different from those of water. A lipid bilayer is ~60 .4 thick, and is non-polar over little more than 30 8, corresponding to the fatty acid chains. Thus, membrane proteins are embedded in a 30 .&-deep non-polar lipid “solvent”. The rest of their surface is in contact with the polar head groups of the lipids or with water on either side of the bilayer. For an integral membrane protein such as bacteriorhodopsin [ 251, which crosses the bilayer, about half of a total accessible surface of some 9000 A2 is estimated to be in a non-polar environment (exact numbers will be available when the detailed three-dimensional structure is known from electron diffraction studies).’ This fraction of the protein surface must be non-polar. Assuming that the other half, in contact with lipid head groups or with water, has the same average properties as for soluble proteins, then 70430% of the bacteriorhodopsin surface rather than the usual 50-60% must be non-polar. This is reflected in its amino-acid composition, typical of integral membrane proteins, with long stretches of non-polar residues. Bacteriorhodopsin folds into ol-helices which pack in the usual ways [ 261, and the “inside” of this protein is not expected to be more polar than that of globular proteins, except where required by its function as a proton channel. Thus, the author does not consider the ‘“inverted protein” structure proposed for membrane proteins to be significant:A specific feature of membrane proteins is the non-polar ring on their surface, not their polar inside. In bacteriorhodopsin, the ring circles the whole protein. In others, it may form the surface of a single a-helix, or of a pair of cu-helices if we believe models [27, 281, which have been proposed for the folding or hydrophobic “signal peptides” that help excreted proteins to pass through membranes while they are synthesized
[=I. CONCLUSIONS
Water is the native environment of most proteins. It affects their structure through the direct and indirect effects analyzed here. It is also involved in function, for instance as a substrate in many enzymatic reactions. The same factors, polar interactions and the hydrophobic effect, which are important in stabilizing proteins, are also active iii promoting ligand binding to active sites, and in building elaborate multi-molecular structures which, like muscle fibres, may reach macroscopic dimensions.
REFERENCES 1 2
C. Tanford, Adv. Protein Chem., 23 (1968) 121. T.E. Creighton, Prog. Biophys. Mol. Biol., 33 (1979)
231-297.
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
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