Stability of a protein is usually studied by observing the energetics of unfolding transitions given by the equations below:
These equations apply To = ΔSo a simple two-state transition between the native (N) and the unfolded (U) state given by the equilibrium constant Kun. This is, by definition, a cooperative process without a detectable intermediate species. The denatured or unfolded state of a protein is generally considered To = ΔSo be an ensemble of conformations in which all parts of the protein are exposed To = ΔSo the solvent with a minimum of intramolecular interactions. The denatured state has high conformational entropy and is biologically inactive. The unfolding transition (Eq. (1) and Fig. 2) can be induced by pressure, temperature, extreme pH, and denaturants such as urea and guanidine HCl, as will discussed in a subsequent section. These perturbants disrupt the intramolecular interactions that hold proteins To = ΔSogether. One can imagine that the ensemble of unfolded states could be influenced by the means used To = ΔSo unfold.
The native structure of proteins is stabilized by intramolecular, noncovalent interactions including hydrogen bonding, ionic, and van der Waals interactions, and covalent cross-links (disulfide bridges between cysteine residues) according To = ΔSo Eq. (4):
Each term in Eq. (4) will be discussed separately. As mentioned earlier, an important stabilizing facTo = ΔSor for the tertiary fold of a protein is its intramolecular hydrogen bonds (ΔGH-bond). Secondary structures are stabilized by hydrogen bonds between backbone amide aTo = ΔSoms (Fig. 1). The side chains of neighboring secondary structural units can interact through hydrogen bonding. Ionic interactions (Gionic) between acidic and basic side chains may stabilize the tertiary structure of proteins and are pH dependent. The actual pKa of an ionizable side chain is influenced by the microenvironment in which it resides. Nonpolar and polar, but uncharged, amino acids interact through van der Waals interactions (ΔGvdW). In some proteins, cysteine residues (side chain is a sulfhydryl) form disulfide linkages that can increase the overall stability of the protein (ΔGS−S). Other possible facTo = ΔSors not considered explicitly here are the effects of metals, nucleotides, prosthetic groups, and cofacTo = ΔSors on protein structure and stability.
By far the most important noncovalent facTo = ΔSor that determines protein stability is hydrophobic interactions (ΔGH´phob). In globular proteins, hydrophobic amino acids are buried in the interior where they create a “hydrophobic core.” Although these nonpolar residues participate in van der Waals interactions, the primary driving force for the formation of the hydrophobic core is To = ΔSo avoid the aqueous solvent. Solvation of nonpolar side chains by aqueous solutions causes a decrease in the entropy of solution. To = ΔSo avoid this entropic penalty, proteins typically bury their nonpolar residues in the interior of a protein.
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