Protein structures, disulfide bonding

Cysteine disulfide formation is one of the most important posttranslational modifications involved in protein structure. Disulfides play a crucial role in maintaining the structure of many proteins including insulin, keratin, and many other structurally important proteins. While the cytoplasm and nucleus are reducing microenvironments, the Golgi and other organelles can have oxidizing environments and process proteins to contain disulfide bonds (Scheme 5). 

Cysteine contains sulfur and can form disulfide bonds to stabilize the shape (tertiary structure) of proteins. Destroying disulfide bonds denatures proteins. 

Spectra, but, in general, leaves the copper site the most exposed of the four cupredoxins. The sequence of Cbp is quite similar to that of stella-cyanin. Stellacyanin is a plant protein, also of unknown function, having visible spectra characteristic of type I copper, but lacking the methionine ligand found in all other type I proteins. A disulfide bond has been suggested as a potential copper ligand in stellacyanin the Cbp has both a methionine and the disulfide, so that prior to the structure determina-... 

Disulfide bond exchange. Disulfide linkages are important in determining protein tertiary structure. Disulfide bond formation and/or exchange may occur during metal-catalyzed oxidation of the cysteine residue. This may lead to protein aggregation due to the formation of intermo-lecular disulfide bonds. In addition to cysteine disulfide bond formation, cysteine is susceptible to oxidation (Fig. 134) (200) (See also discussion on thiol chemistry earlier in this chapter). 

Disulfide bonds These covalent bonds form between Cys residues that are close together in the final conformation of the protein (see Fig. 4) and function to stabilize its three-dimensional structure. Disulfide bonds are really only formed in the oxidizing environment of the endoplasmic reticulum (see Topic A2), and thus are found primarily in extracellular and secreted proteins. 

The dehydron/disulfide balance relation clearly identifies proteins with excess (Y > 5X + 20) or lack (Y < 5X + 20) of structural deficiencies, with the former likely to be more favorably denatured than the latter under equivalent redox and denatura-tion conditions. To test this prediction, thermodynamic data on thermal denaturation (Table 2.2) were obtained for an exhaustive set of proteins for which structural information was also available [7], Thus, the thermal denaturation free energy change, AG, under reducing conditions and comparable temperatures [11], was obtained for monomeric uncomplexed PDB-reported proteins with disulfide bonds and lacking prosthetic groups or ion coordination. A significant anticorrelation was found... 

The folding of proteins into their three-dimensional structure is essential for their biological function. For proteins that contain disulfide bonds, formation of these bonds is often an important step in the folding reaction. The presence of one or more disulfide bonds is crucial to the maintenance of the folded state of many secretory proteins. In contrast, cytosolic proteins form disulfide bonds only as part of their catalytic cycle, and are not stabilized by these bonds. 

In extracellular proteins, the oxidizing environment leads to formation of disulfide bonds whenever two free cysteine side chains can assume the appropriate configuration. Not surprisingly, the range of tertiary structures in proteins with disulfide bonds is more varied than that normally observed for intracellular proteins. 

Observed Structure in Proteins without Disulfide Bonds... 

The shape of a large protein is influenced by many factors including of course Its primary and secondary structure The disulfide bond shown m Figure 27 18 links Cys 138 of carboxypeptidase A to Cys 161 and contributes to the tertiary structure Car boxypeptidase A contains a Zn " ion which is essential to the catalytic activity of the enzyme and its presence influences the tertiary structure The Zn ion lies near the cen ter of the enzyme where it is coordinated to the imidazole nitrogens of two histidine residues (His 69 His 196) and to the carboxylate side chain of Glu 72... 

The primary structure of a peptide is given by its ammo acid sequence plus any disulfide bonds between two cysteine residues The primary structure is determined by a systematic approach m which the protein is cleaved to smaller fragments even individual ammo acids The smaller fragments are sequenced and the mam sequence deduced by finding regions of overlap among the smaller peptides... 

A prior distribution for sequence profiles can be derived from mixtures of Dirichlet distributions [16,51-54]. The idea is simple Each position in a multiple alignment represents one of a limited number of possible distributions that reflect the important physical forces that determine protein structure and function. In certain core positions, we expect to get a distribution restricted to Val, He, Met, and Leu. Other core positions may include these amino acids plus the large hydrophobic aromatic amino acids Phe and Trp. There will also be positions that are completely conserved, including catalytic residues (often Lys, GIu, Asp, Arg, Ser, and other polar amino acids) and Gly and Pro residues that are important in achieving certain backbone conformations in coil regions. Cys residues that form disulfide bonds or coordinate metal ions are also usually well conserved. 

Disulfide bonds in proteins are generally stable and nonreactive, acting like bolts in the structure. However, oxidized DsbA is less stable than the reduced form and its disulfide bond is very reactive. DsbA is thus a strong... 

Figure 6.8 Schematic diagram of the enzyme DsbA which catalyzes disulfide bond formation and rearrangement. The enzyme is folded into two domains, one domain comprising five a helices (green) and a second domain which has a structure similar to the disulfide-containing redox protein thioredoxin (violet). The N-terminal extension (blue) is not present in thioredoxin. (Adapted from J.L. Martin et al.. Nature 365 464-468, 1993.)...

Figure 15.18 (a) Schematic representation of the path of the polypeptide chain in the structure of the class I MHC protein HLA-A2. Disulfide bonds are indicated as two connected spheres. The molecule is shown with the membrane proximal immunoglobulin-like domains (a3 and Pzm) at the bottom and the polymorphic al and a2 domains at the top. 

In addition to being a remarkable demonstration of the power of computer-based combinatorial design of a protein fold, this designed peptide is the shortest known peptide consisting entirely of naturally occurring amino acids that folds into a well-ordered structure without metal binding, oligomerization or disulfide bond formation. 

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