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The General Principles of Protein Folding
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Proteins are the fundamental biopolymer responsible for much of the biochemistry of living systems. The remarkable versatility of proteins often results from their capacity to spontaneously fold into a defined conformation with specific biological or chemical function. Dysregulation of folding can result in a consequent metabolic dysregulation to give rise to debilitating pathological states. The design of synthetic proteins holds the potential to exploit their functional capacity for novel purposes, although much work remains to achieve a complete understanding of protein folding.
Proteins (polypeptides) are one of the three principle linear, unbranched biopolymers that define living systems, along with polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA). Although all three classes of biopolymers are rich in chemical information, it is proteins that serve as the key structural components, molecular machines, and catalysts that enable complex metabolism. Proteins are unique compared to other biopolymers because they must often ‘fold’ into a precise ensemble of closely related conformations, the ‘native state,’ to be functionally active. The structure of the native state is predominantly defined by the amino acid sequence of the protein, but can be modulated by environmental factors (e.g., temperature, pH, ionic strength, ligand concentration, voltage across a membrane, or mechanical stress) with functional consequences. To date, an analytical understanding of how amino acid sequence and chemical environment shape the native state structure remains elusive, though many general principles of protein structure and folding are known.
Protein Stability, A Delicate Balance between Enthalpy and Entropy
To a first approximation, the folding of a protein can be viewed as a transition between two thermodynamic states: the native state (an ensemble of closely related, compact conformations) and the ‘denatured state’ (a heterogeneous ensemble of extended, flexible conformations). The native state is most often associated with biological function whereas the denatured state tends to be nonfunctional, susceptible to degradation by proteases, and prone to aggregation. The overall stability of a protein – that is, the energetic difference between the native state and the denatured state – can be thought of as a ‘tug of war’ between various entropic and enthalpic considerations that favor one state over the other. In general, the native state is only marginally more stable than the denatured state, with an energy gap between the two states equivalent in magnitude to only a few hydrogen bonds.
The flexibility of the denatured state is a direct consequence of the molecular structure of the protein backbone, which traces the covalent bonds that ‘string together’ amino acids to form a polypeptide. For each peptide bond (the chemical link between amino acids) two freely-rotatable, single bonds are present in the protein backbone. In the denatured state, rotation about these single bonds is unimpeded and a vast number of conformations can be sampled. Upon folding, the conformational freedom of the backbone is constrained by the reinforcing intramolecular interactions that comprise the native state. Thus, from the perspective of the polypeptide chain, collapse into the native state structure is accompanied by a dramatic ‘loss of entropy’ (an unfavorable transition).
The native state and the denatured state make fundamentally different interactions with the surrounding solvent (water molecules) and, as a consequence, solvent structure must be considered to understand protein stability and folding. In the denatured state, nearly all residues – including both hydrophilic and hydrophobic side-chains – are solvent-exposed; thus, hydrogen bond donors and acceptors that reside on the protein backbone and side-chains are satisfied by water. The hydrophobic moieties of a denatured protein, however, are unable to form hydrogen bonds and, instead, induce a structural transition in the adjacent water molecules. To avoid the loss of a hydrogen bond (an unfavorable loss of enthalpy) water molecules form an ice-like cage, referred to as a ‘clathrate,’ around the hydrophobic group. The clathrate structure, however, is itself a trade-off by maximizing the number of hydrogen bonding partners, clathrate waters suffer a substantial loss of translational and rotational freedom (an unfavorable loss of entropy). The formation of clathrates is the basis of the ‘hydrophobic effect’ and is a major driving force for protein folding.
Upon folding into the native structure, the hydrophobic residues that were exposed to solvent in the denatured state are buried into a solvent-excluded core, thereby releasing the clathrate waters. In addition, the hydrogen bonding of backbone donors and acceptors becomes largely self-satisfied by the native state structure, which serves as the basis for the commonly observed types of protein 2° structure (e.g., α-helix or β-strand). Thus, a large number of the waters that were H-bonded to polar groups in the denatured state are released when the protein adopts the native state structure. Taken together, collapse into the native state, from the perspective of the solvent, is accompanied by a dramatic ‘gain of entropy’ (a favorable transition).
In addition to the entropic benefit of sequestering hydrophobic residues into a solvent-excluded core, protein core formation is associated with favorable London dispersion forces, resulting in an enthalpic contribution to folding. To maximize the enthalpy of packing, protein cores often exhibit features of structural complementarity with minimal packing defects, resembling a sort of three-dimensional jigsaw puzzle. Other enthalpic contributions that favor folding result from ‘salt bridges,’ which are combined hydrogen bonding and electrostatic interactions between oppositely charged, hydrogen bonding-competent side-chains (e.g., lysine and glutamic acid).
Protein stability involves complex trade-offs between the configurational entropy of the protein backbone, the entropy of the solvent, and the favorable enthalpies of interaction between protein groups, such as backbone hydrogen-bonding within 2° structure, salt bridges, and London dispersion forces in the core (Figure 1). Enumeration of these terms typically identifies large opposing energetic contributions between enthalpy and entropy (e.g., |ΔH| and |-TΔS| of folding are often >400 kJ mol−1 for small proteins) but with a small net balance (e.g., 20–30 kJ mol−1) in favor of the native state structure. Thus, evolved proteins have a modest energetic preference for the folded conformation – a balance that can, in some cases, be disrupted by a single energetically unfavorable point mutation. For example, a single strong salt bridge can provide 10–15 kJ mol−1 of favorable stability; thus, a mutation that eliminates one member of the salt bridge can cause protein unfolding (Anderson et al., 1990).
To read more about protein folding, misfolding and disordered proteins along with related diseases, click here.
This excerpt was taken from the article Proteins: Folding, Misfolding, Disordered Proteins, and Related Diseases from the recently published Encyclopedia of Cell Biology. This important work includes over 300 articles covering every aspect of cell biology, with fully annotated figures, abundant illustrations and references for further reading enabling you to advance your research.
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