Disulfide structure proteins

In addition to classification based on layer structure, proteins can be grouped according to the type and arrangement of secondary structure. There are four such broad groups antiparallel a-helix, parallel or mixed /3-sheet, antiparallel /3-sheet, and the small metal- and disulfide-rich proteins. 

One of the most convenient ways of generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. 

Although the occurrence of six conserved cysteine residues, the spacing patterns of these residues, and possibly the pattern of disulfide structures are hallmarks of OBPs, the six-cysteine criterion alone is not sufficient to classify a certain protein as an olfactory protein [ 16]. It is important to demonstrate that an OBP is expressed only (or predominantly) in olfactory tissues. Evidence for their ability to bind odorants is also desirable, but not sine qua non. One of these criteria alone would not be enough to define a given protein as an OBP. For example, bovine serum albumin (BSA) binds to insect pheromones (Leal, unpublished data) and yet it is not an OBP because it not expressed in insect olfactory tissues. Conversely, a protein specific to antennae is not necessarily an OBP. There are other proteins that may be expressed in antennae but not in control tissues. Non-OBPs specifically accumulated in insect antennae have been previously detected (Ishida and Leal, unpublished data). Also, a glu-tathione-S-transferase has been reported to be expressed specifically in antennae of M. sexta [52]. 

The dihedral angles of disulfides in proteins are very difficult to determine with any accuracy except in refined high-resolution structures. In the first few protein structures to show disulfides at 2 A resolution, attention was paid mostly to the dihedral angle around the S—S bond (x3), since it is the most characteristically interesting... 

Fig. 47. The Xi angles observed for disulfides in protein structures. The examples from refined, high-resolution structures are shown separately at the top.

Now let us examine the distribution and position of disulfides in proteins. The simplest consideration is distribution in the sequence (see Fig. 51), which is apparently quite random, except that there must be at least two residues in between connected half-cystines. Even rather conspicuous patterns such as two consecutive halfcystines in separate disulfides turn out, when the distribution is plotted for the solved structures (Fig. 51), to occur at only about the random expected frequency. The sequence distribution of halfcystines is influenced by the statistics of close contacts in the three-dimensional structures, but apparently there are no strong preferences of the cystines that could influence the three-dimensional structure. 

One erf the most convenient ways erf generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. In some cases, mild reducing conditions can free one or more —SH groups for conjugation or modification purposes. The creation erf free sulf-hydryls in this manner allows for site-directed modification at a limited number of locations within the protein molecule. 

Leal W. S., Nikonova L. and Peng G. (1999) Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori. FEBS Letters. 468, 85-90. 

Proteins spontaneously fold into their native conformation, with the primary structure of the protein dictating its three-dimensional structure. Protein folding is driven primarily by hydrophobic forces and proceeds through an ordered set of pathways. Accessory proteins, including protein disulfide isomerases, peptidyl prolyl cis-trans isomerases, and molecular chaperones, assist proteins to fold correctly in the cell. 

Disulfides. Relative to the aromatic chromophores in proteins, the CD spectra of disulfides in proteins has received little attention. This is due to a number of reasons. First, the signals from the disulfides are typically weaker than those arising from aromatic groups. Second, the nature of higher energy excited states in disulfides is poorly understood. Third, little data is available on their electronic structure, either from theoretical treatments [114-116] or from experimental studies [117],... 

Susceptibility to oxidation of disulfides built into proteins is strongly dependent on their location in the protein molecule (G3). Since the disulfides have a crucial role in maintaining protein tertiary structure, oxidation of certain —S—S— bridges may expose further disulfides and cause unfolding of the protein molcule. The final disulfide oxidation is a sulfone residue, which is stable and does not tend to reverse to sulfide. Therefore oxidative breakage of disulfides is irreversible. The spatial location of disulfides inside protein molecules influences their susceptibility to oxidation. The ribonuclease molecule has four —S—S— bonds, and at least three correctly located disulfide bonds are necessary to retain the ribonuclease enzyme properties. The compact ribonuclease molecule is relatively resistant to HOC1 oxidation (D18). 

The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGF. This increased mobility arises from the fact that, as disulfide-bonded proteins are intra-molecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Gold-enberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGF. The translocated protein forms disulHde bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. 

The lack of reversibility, and the presence of precipitation, makes thermodynamic analysis of aFGF particularly challenging. Precipitation in the presence of DTT indicates that precipitation is not dependent upon the formation of mixed disulfides. Structural analysis of human aFGF (17) shows that the three free cysteine residues are located at solvent inaccessible positions (figure 4). Thus, formation of mixed disulfides would be expected to destabilize the protein because a) structural changes would be required to expose the cysteines for oxidation and b) covalent adducts of the cysteine residues would have to be tolerated within the packing constraints of the interior of the protein for the native state to be adopted. 

Proteins are characterized by their primary, secondary, tertiary, and quaternary structures. The primary structure is the sequence of the amino acids in the polypeptide chain that makes up the protein. Secondary structure refers to the hrst folding of the amino acid chain and reflects, for example, disulfide bonds. Tertiary structure (a monomer) is the final folded configuration of the protein that is controlled by the primary and secondary structures and is thermodynamically driven by the relative hydrophobicity of the component amino acids in the structure. Quaternary structure refers to the functional association of several polypeptides (monomers). For example, the final structure of hemoglobin consists of four associated monomers. Any change in the primary structure of a protein often results in changes to aU the higher level structure. Protein structures must be characterized and controlled during the production process. 

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