Protein disulfide exchange

E, and K which must be assembled in the correct sequence. A chaperonin PapD is also required as is an "usher protein," PapC,50 and also the disulfide exchange protein DsbA (Chapter 10). DsbA helps PapD to form the correct disulfide bridges as it folds and PapD binds and protects the various pilus subunits as they accumulate in the periplasmic space of the host. The usher protein displaces the chaperonin PapD and "escorts" the subunits into the membrane where the extrusion occurs.50 55... 

Disulfide bonds between and within polypeptides stabilize tertiary and quaternary structure. However, disulfide bond formation is nonspecific. Under oxidizing conditions, a given cysteine can form a disulfide bond with the —SH of any accessible cysteinyl residue. By catalyzing disulfide exchange, the rupture of an S— bond and its reformation with a different partner cysteine, protein disulfide isomerase facilitates the formation of disulfide bonds that stabilize their native conformation. 

Disruption of the native structure of a protein can also contribute to chemical instability by accelerating the rates of a variety of degradation routes, including deamidation, hydrolysis, oxidation, disulfide exchange, /1-elimination, and racemization. 

Disulfide exchange reactions occur over a broad range of conditions—from acid to basic pH—and in a wide variety of buffer constituents. Most crosslinking reactions involving disulfide exchange are done under physiological conditions or those most appropriate to maintain stability of the protein or other molecule being modified. 

Fig. 7. Kinetics of return of native antigenic structure in various limited regions of reduced albumin as determined with restricted populations of antialbumin. Reduced protein was reoxidized in the presence of optimal amounts of rat liver disulfide-exchange enzyme (0.5 mg/ml of RII/2.5 ml of reaction mixture). The reoxidation buffer and other conditions were as described in Section II. (A) 0, Anti-T 5 1M , anti- Tstt-mi anti-N-fragment antialbumin, anti-C-ffagment and anti anti-T,M, j.

Post-synthetic functionalization reactions have been reported that make it possible to introduce new groups into folded proteins and peptides using natural [24, 25] as well as non-natural amino acids [26-30]. The disulfide exchange... 

Disulfide bond formation was introduced into DCC as a powerful reaction for the construction of dynamic systems in the late 1990s in separate reports from the groups of Still [19], Sanders [20], and Lehn [21]. Given the fundamental role played by thiol oxidation in biology, it is no surprise that the reaction is highly compatible with protein targets. Disulfide exchange... 

The DCL was created under standard disulfide exchange conditions at pH 7.5. After equilibration for 48 hours, each of the expected 15 disulfide products could be observed in the library using LC-MS analysis (Fig. 2.11). The library was then equilibrated in the presence of CaM, followed by a centrifugation filtration step to separate protein/bound components from free components in solution. Analysis of the filtrate was complicated by the filtration membrane affecting the composition of the library. The bound components, however, provided meaningful results. Denaturation and filtration afforded a mixture of all peptides that had bound to CaM in the course of the DCL. HPLC analysis indicated significant amplification of dimer cc (80%) and a small amplification of dimer ec (10%). Resynthesis of these two components and binding assay established values of 10 and... 

Drugs coupled via a disulfide bond like, captopril, are rapidly released from the protein-spacer moiety of the conjugate, enzymatically by 3-lyase and/or non-enzymatically by thiol-disulfide exchange with endogenous thiols [68]. 

All proteins and peptides display chemical and physical instability that affects the way they are distributed and cleared in the body and their delivery to the site of action. Physical and chemical instability is affected by primary sequences and secondary and tertiary structures and the degree of glyco-sylation of protein. Chemical degradation of proteins and peptides involves deamidation, racemization, hydrolysis, oxidation, beta elimination, and disulfide exchange. Physical degradation of proteins involves denaturation and aggregation. 

Proteins, peptides, and other polymeric macromolecules display varying degrees of chemical and physical stability. The degree of stability of these macromolecules influence the way they are manufactured, distributed, and administered. Chemical stability refers to how readily the molecule can undergo chemical reactions that modify specific amino-acid residues, the building blocks of the proteins and peptides. Chemical instability mechanisms of proteins and peptides include hydrolysis, deamidation, racemization, beta-elimination, disulfide exchange, and oxidation. Physical stability refers to how readily the molecule loses its tertiary and/or sec-... 

The first natural sulfhydryl activator of FDPase to be described was cystamine, which at pH 7.5 was found to undergo a disulfide exchange reaction with 2 reactive cysteine residues in the protein (45), leading... 

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