Protein structure, metal stabilization

The native conformation of proteins is stabilized by a number of different interactions. Among these, only the disulfide bonds (B) represent covalent bonds. Hydrogen bonds, which can form inside secondary structures, as well as between more distant residues, are involved in all proteins (see p. 6). Many proteins are also stabilized by complex formation with metal ions (see pp. 76, 342, and 378, for example). The hydrophobic effect is particularly important for protein stability. In globular proteins, most hydrophobic amino acid residues are arranged in the interior of the structure in the native conformation, while the polar amino acids are mainly found on the surface (see pp. 28, 76). 

Zinc in metalloenzymes may (i) participate directly in the catalytic process, (ii) serve to stabilize protein structure or (iii) have a regulatory role. In each case, removal of the metal from the holoenzyme generally results in an apoprotein having no catalytic activity. The enzymes considered briefly below provide examples of each of these functions of Zn. The study of zinc metalloproteins has often in the past been beset by analytical problems and by contamination with traces of metal ions a review covering these important topics has appeared.1263 Another recent review deals with the physiological, nutritional and medical role of zinc.1264... 

Phospholipase C from B. cereus contains two zinc ions per molecule of protein (molecular weight 23 000), the two metal ions being about 5 A apart. The zinc appears to have a particular role in the stabilization of the protein structure.585 Added Zn11 protects the enzyme against denaturation and induces the refolding of the denatured enzyme. Other metals are much less effective than zinc in carrying out this function.586... 

The calcium ion in a-LA plays a structural role in stabilizing the protein. The thermal stability of the calcium-bound form of a-LA increases more than 40 °C compared to that of the apo-form. At low pH (e.g. pH 2), a-LA releases the calcium ion and becomes partially unfolded (molten globule state). This partially unfolded protein loses its tertiary structure but retains its secondary structure. Other metals, such as manganese or magnesium, are able to compete with calcium at the same site with a similar stabilizing effect. However, the binding of zinc, which is proposed to bind at different locations, decreases a-LA stability. ... 

A fundamental premise of the entatic state concept is that the metal simply fits into a site that is preformed by the protein and determined by the many interactions which stabilize the protein structure. This idea has been tested by crystallographic studies of apo-forms of azurin, plastocyanin, pseudoazurin and amicyanin. 

B. Redesign of Nonheme Iron Proteins. In heme protein redesign described above, the heme prosthetic group largely dictates the active site structure. Redesign focuses mainly on the proximal and distal sides of the heme, causing minimal effects on the overall protein scaffolds. This is not necessarily the case for nonheme metalloproteins in which metal sites are not as dominant and small changes may have more dramatic effects on the protein folds and stability. 

Metalloproteins constitute a distinct subclass of proteins that are characterized by the presence of single or multiple metal ions bound to the protein by interactions with nitrogen, sulfur, or oxygen atoms of available amino acid residues or are complexed by prosthetic groups, such as heme, that are covalently linked to the protein. These metals function either as catalysts for chemical reactions or as stabilizers of the protein tertiary structure. Protein-bound metals may also be labile and, as such, be subject to transport, transient storage, and donation to other molecular sites of requirement within tissues and cells. 

Many enzymes contain metal ions as an integral part of their structures (e.g., zinc in ALP and carboxypeptidase A). The function of the metal may be to stabilize tertiary and quaternary protein structures. Removal of divalent metal ions by treatment with an appropriate concentration of EDTA solution is accompanied by conformational changes with inactivation of the enzyme. The enzyme can often be reactivated by dialysis against a solution of the appropriate metal ion or simply by adding the ion to the reaction mixture. Reactivation may take some time, because rearrangement of the polypeptide chains into the active conformation is not instantaneous. 

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