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FIGURE 7.49 Zinc Finger DNA-Binding Protein

A central zinc atom is bound to the sulfurs of cysteine (C) and the nitrogens of histidine (H). Chains of amino acids of varying lengths (x = chain length) extend from these binding regions. The zinc finger forms a component of a much larger protein and binds the protein to DNA.

The correct 3D structure of proteins can be destroyed by heating, detergents, acids, bases and certain chemicals.

many of them have multiple fingers. The first zinc finger protein discovered was the general eukaryotic transcription factor TFIIIA, from the toad Xenopus, which has nine zinc fingers.

Each zinc finger unit usually recognizes three bases in the DNA. Less often, four or five bases are recognized by a single zinc finger. The sequence specificity of each zinc finger depends on the amino acid sequence of the polypeptide chain between the His and Cys residues that bind the zinc. Amino acids in this region make hydrogen bonds with bases in the DNA.

Several modified versions of the zinc finger motif have been found. For example, the steroid receptor family of transcription factors has a DNA-binding domain that contains two Zn atoms, each surrounded by four cysteines. A short a-helix that lies between the two zincs binds to the DNA. Several fungal transcription factors, including GAL4 of yeast, contain fingers built around a cluster of two Zn atoms bound to six cysteines.

Denaturation of Proteins

Denaturation is the loss of correct 3-D structure, i.e. the loss of both tertiary and quaternary structure. Denaturation only involves the breaking of non-covalent bonds. Loss of biological activity generally accompanies such structural denaturation. In particular, enzymes are especially sensitive to denaturation.When proteins are denatured they often precipitate out of solution, as happens to the proteins of an egg when it is boiled. Heat, extremes of pH, and a variety of chemical agents destroy non-covalent structure and denature proteins. Even agitation may denature some proteins as when egg whites are whipped to give meringues.

Proteins vary greatly in their stability. Some are very sensitive and even slight changes in pH or temperature may inactivate them. This often causes problems in both the purification of proteins and their biotechnological applications. Consequently, researchers have often searched for natural proteins that are unusually resistant, or have modified proteins to increase their stability. Bacteria that live under extreme conditions are a rich source of such resistant proteins. For example, the Taq polymerase denaturation Loss of correct 3D structure of proteins or nucleic acids steroid receptor Protein that binds steroid hormones

Denaturation of Proteins 195

FIGURE 7.50 Structure and Function of Sodium Dodecyl Sulfate (SDS)

A) SDS contains a strongly hydrophobic carbon chain and a strongly hydrophilic sodium sulfate region. B) When a folded protein is boiled in the presence of SDS, the hydrophobic region winds around the polypeptide backbone and the negatively charged sulfate protrudes. The negative charges repel each other which helps in straightening out the protein.

Denaturation of Proteins 195

FIGURE 7.50 Structure and Function of Sodium Dodecyl Sulfate (SDS)

A) SDS contains a strongly hydrophobic carbon chain and a strongly hydrophilic sodium sulfate region. B) When a folded protein is boiled in the presence of SDS, the hydrophobic region winds around the polypeptide backbone and the negatively charged sulfate protrudes. The negative charges repel each other which helps in straightening out the protein.

Detergents and chaotropic agents help solubilize hydrophobic groups in water.

Sodium dodecyl su Ifate is widely used to solubilize proteins before running them on gels.

Chaotropes alter the structure of water, so allowing hydrophobic groups to dissolve more easily.

used in PCR (see Ch. 23) is a highly heat-resistant enzyme made by bacteria that live naturally at temperatures so high they would kill most organisms.

The hydrophobic forces responsible for maintaining much of the 3-D structure of proteins are disrupted by detergents and chaotropic agents. Detergents consist of a hydrophobic tail joined to a highly water-soluble group. They act by directly binding to the hydrophobic regions of other molecules and solubilizing them. In the case of proteins, detergents can bind to the hydrophobic groups that are normally buried deep inside and also to the relatively hydrophobic polypeptide backbone. This destabilizes the 3-D folding of the polypeptide chain.

The detergent sodium dodecyl sulfate (SDS) is widely used to solubilize and denature proteins before running them on polyacrylamide gels to separate them by molecular weight (see Ch. 26). SDS has a long hydrocarbon tail that binds to the polypeptide and a negatively charged sulfate group that sticks out into the water and solubilizes the protein/SDS complex (Fig. 7.50). SDS binds to polypeptides along their long axes and converts them to an extended rod-shaped conformation. The precise nature of SDS-polypeptide binding is disputed. One theory is that it binds to the non-polar R-groups of hydrophobic amino acids. However, SDS binds in a ratio of one SDS to every two amino acid residues for the vast majority of proteins and this suggests that it binds to the polypeptide backbone. From a practical viewpoint, it is important that the number of negative charges contributed by bound SDS is proportional to the length of the polypeptide, i.e. to its molecular weight.

Chaotropic agents (e.g. thiocyanate, perchlorate) also destabilize proteins by promoting the exposure of hydrophobic groups, but by an indirect mechanism. When exposed to water, hydrophobic groups induce the formation of regular cages of water molecules around themselves. This decreases the disorder of the water; i.e., it causes an increase in entropy, which is thermodynamically unfavorable. Hydrophobic groups tend to cluster together to avoid contact with water, rather than because of any positive attraction. Chaotropes disrupt water structure and so allow hydrophobic groups to dissolve more readily.

chaotropic agent Chemical compound that disrupts water structure and so helps hydrophobic groups to dissolve detergent Molecule with both hydrophobic and hydrophilic regions that can solubilize hydrophobic molecules including fats, grease and lipids sodium dodecyl sulfate (SDS) A detergent widely used to denature and solubilize proteins before separation by electrophoresis

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