Improving Stability in Other Ways

IMPROVING STABILITY IN OTHER WAYS

Although the introduction of disulfide bonds is the simplest and most effective way to  increase protein stability, a variety of other alterations may also help. Because of the effects of entropy, the greater the number of possible unfolded conformations, the more likely a protein is to unfold. Decreasing the number of possible unfolded conformations therefore promotes stability. This may be done in several ways, in addition to introducing extra disulfide linkages as described earlier. Glycine, whose R-group is just a hydrogen atom, has more conformational freedom than any other amino acid residue. In contrast, proline,  with its rigid ring, has the least conformational freedom. Therefore replacing glycine with any other amino acid or increasing the number of proline residues in a polypeptide chain will increase stability. Such replacements must avoid altering the structure of the protein, especially in critical regions. When tested experimentally, such changes do contribute small increases in stability.

 Because hydrophobic residues tend to exclude water, these residues tend to cluster in the center of proteins and avoid the outer surface. If cavities exist in the hydrophobic core, filling them should increase protein stability. This may be done by replacing small hydrophobic residues, which are already in or near the core, with larger ones. For example, changing Ala to Val or Leu to Phe will achieve this. However, most proteins have hydrophobic cores that are already fairly stable and have few cavities. Furthermore, inserting larger hydrophobic amino acids to fill these cavities often causes twisting of their side chains into unfavorable conformations. This cancels out any gains of stability from packing the hydrophobic core more completely.

 Because of its asymmetrical structure, the alpha helix is actually a dipole with a slight positive charge at its N-terminal end and a slight negative charge at its C-terminal end. The presence of amino acid residues with the corresponding opposite charge close to the ends of an alpha helix promotes stability. In natural proteins the majority of alpha helixes are stabilized in this manner. However, in cases where such stabilizing residues are absent, protein engineering may create them.

 Asparagine and glutamine residues are relatively unstable. High temperature or extremes of pH convert these amides to their corresponding acids, aspartic acid and glutamic acid. The replacement of the neutral amide by the negatively charged carboxyl may damage the structure or activity of the protein. This may be avoided by engineering proteins to replace Asn or Gln by an uncharged hydrophilic residue of comparable size, such as Thr.