Protein stability and folding

Upon biosynthesis, a polypeptide folds into its native conformation, which is structurally stable and functionally active. The conformation adopted ultimately depends upon the polypeptide's amino acid sequence, explaining why different polypeptide types have different characteristic conformations. We have previously noted that stretches of secondary structure are stabilized by short-range interactions between adjacent amino acid residues. Tertiary structure, on the other hand, is stabilized by interactions between amino acid residues that may be far apart from each other in terms of amino acid sequence, but which are brought into close proximity by protein folding. The major stabilizing forces of a polypeptide's overall conformation are:

• hydrophobic interactions

• electrostatic attractions

• covalent linkages.

Hydrophobic interactions are the single most important stabilizing influence of protein native structure. The 'hydrophobic effect' refers to the tendency of non-polar substances to minimize contact with a polar solvent such as water. Non-polar amino acid residues constitute a significant proportion of the primary sequence of virtually all polypeptides. These polypeptides will fold in such a way as to maximize the number of such non-polar residue side chains buried in the polypeptide's interior, i.e. away from the surrounding aqueous environment. This situation is most energetically favourable.

Stabilizing electrostatic interactions include van der Waals forces (which are relatively weak), hydrogen bonds and ionic interactions. Although nowhere near as strong as covalent linkages (Table 2.5), the large number of such interactions existing within a polypeptide renders them collectively quite strong.

Although polypeptides display extensive networks of intramolecular hydrogen bonds, such bonds do not contribute very significantly to overall conformational stability. This is because atoms hydrogen bonding with each other in a folded polypeptide can form energetically equivalent hydrogen bonds with water molecules if the polypeptide is in the unfolded state. Ionic attractions between (oppositely) charged amino acid side chains also contribute modestly to overall protein conformational stability. Such linkages are termed salt bridges, and, as one would expect, they are located primarily on the polypeptide surface.

Table 2.5 Approximate bond energies associated with various (non-covalent) electrostatic interactions, compared with a carbon-carbon single bond

Bond type

Bond strength (kJ mol 1)

Van der Waals forces

10

Hydrogen bond

20

Ionic interactions

86

Carbon-carbon bond

350

Disulfide bonds represent the major covalent bond type that can help stabilize a polypeptide's native three-dimensional structure. Intracellular proteins, although generally harbouring multiple cysteine residues, rarely form disulfide linkages, due to the reducing environment that prevails within the cell. Extracellular proteins, in contrast, are usually exposed to a more oxidizing environment, conducive to disulfide bond formation. In many cases the reduction (i.e. breaking) of disulfide linkages has little effect upon a polypeptide's native conformation. However, in other cases (particularly disulfide-rich proteins), disruption of this covalent linkage does render the protein less conformationally stable. In these cases the disulfide linkages likely serve to 'lock' functional/structurally important elements of domain/tertiary structure in place.

The description of protein structure as presented thus far may lead to the conclusion that proteins are static, rigid structures. This is not the case. A protein's constituent atoms are constantly in motion, and groups ranging from individual amino acid side chains to entire domains can be displaced via random motion by anything up to approximately 0.2 nm. A protein's conformation, therefore, displays a limited degree of flexibility, and such movement is termed 'breathing'.

Breathing can sometimes be functionally significant by, for example, allowing small molecules to diffuse in/out if the protein's interior. In addition to breathing, some proteins may undergo more marked (usually reversible) conformational changes. Such changes are usually functionally significant. Most often they are induced by biospecific ligand interactions (e.g. binding of a substrate to an enzyme or antigen binding to an antibody).

2.4.1 Structural prediction

Currently, there exists an enormous and growing deficit between the number of polypeptides whose amino acid sequence has been determined and the numbers of polypeptides whose three-dimensional structure has been resolved. Given the complexities of resolving three-dimensional structure experimentally, it is not surprising that scientists are continually attempting to develop methods by which they could predict higher order structure from amino acid sequence data. Although modestly successful secondary structure predictive approaches have been developed, no method by which tertiary structure may be predicted from primary data has thus far been developed.

Over 20 different methods of secondary structure prediction have been reported (Table 2.6). The approaches taken fall into two main categories:

Table 2.6 Some secondary structure predictive methods currently used. Refer to text for further details

Method Basis of prediction

Chou and Fasman Empirical statistical method

Garnier, Osguthorpe and Robson (GOR) Empirical statistical method

EMBL profile neural network (PHD) Empirical statistical method

Protein sequence analysis (PSA) Empirical statistical method

Lim Physicochemical criteria

1. Empirical statistical methods, which are based upon data generated from studying proteins of known three-dimensional structure and correlation of such proteins' primary amino acid sequences with structural features.

2. Methods based upon physicochemical criteria, such as fold compactness (i.e. the generation of a folded form displaying a tightly packed hydrophobic core and a polar surface).

Most such predictive methods are at best 50-70 per cent accurate. The relatively large inaccuracy stems from the fact that the folded (tertiary) structure imposes constraints upon the nature/extent of secondary structure within some regions of the polypeptide chain. Any generalized 'rules' relating secondary structure to amino acid sequence data, by nature, will not take such issues into consideration.

Accurate prediction of a polypeptide's three-dimensional structure from lower order structural information remains to be achieved. Tertiary structure prediction directly from amino acid sequence data remains in the distant future, although a technique known as threading will likely support some progress towards this goal. Three-dimensional structural analysis has shown that only a limited number of stable protein folds exist and, moreover, that many unrelated amino acid sequences can generate the same fold (a fold refers to a domain-like structure that is common to many proteins). By analysing databases containing polypeptide tertiary structure information, the various possible amino acid sequences that can give rise to any particular fold can potentially be determined.

Threading essentially entails comparing the sequence of the polypeptide whose three-dimensional structure you wish to predict with the database sequences known to generate specific fold patterns. Computer programs can then be used to estimate the probability of the target sequence adopting each known folding structure.

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Responses

  • stephen
    What bonding contributes to stability of folded polypeptide?
    7 years ago

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