Disulfide bonds formation

The second application of the CFTI approach described here involves calculations of the free energy differences between conformers of the linear form of the opioid pentapeptide DPDPE in aqueous solution [9, 10]. DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen, where D-Pen is the D isomer of /3,/3-dimethylcysteine) and other opioids are an interesting class of biologically active peptides which exhibit a strong correlation between conformation and affinity and selectivity for different receptors. The cyclic form of DPDPE contains a disulfide bond constraint, and is a highly specific S opioid [llj. Our simulations provide information on the cost of pre-organizing the linear peptide from its stable solution structure to a cyclic-like precursor for disulfide bond formation. Such... 

The Cyc conformer represents the structure adopted by the linear peptide prior to disulfide bond formation, while the two /3-turns are representative stable structures of linear DPDPE. The free energy differences of 4.0 kcal/mol between pc and Cyc, and 6.3 kcal/mol between pE and Cyc, reflect the cost of pre-organizing the linear peptide into a conformation conducive for disulfide bond formation. Such a conformational change is a pre-requisite for the chemical reaction of S-S bond formation to proceed. 

Y. Wang and K. Kuczera. Conformational free energy surface of the linear DPDPE peptide Cost of pre-organization for disulfide bond formation. J. Am. Chem. Soc., submitted, 1997. 

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.)...

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. 

In 2000, the first example of ELP diblock copolymers for reversible stimulus-responsive self-assembly of nanoparticles was reported and their potential use in controlled delivery and release was suggested [87]. Later, these type of diblock copolypeptides were also covalently crossUnked through disulfide bond formation after self-assembly into micellar nanoparticles. In addition, the encapsulation of l-anilinonaphthalene-8-sulfonic acid, a hydrophobic fluorescent dye that fluoresces in hydrophobic enviromnent, was used to investigate the capacity of the micelle for hydrophobic drugs [88]. Fujita et al. replaced the hydrophilic ELP block by a polyaspartic acid chain (D ). They created a set of block copolymers with varying... 

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

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. 

Polyacrylamide gel electrophoresis results suggest that p-LG undergoes a greater conformational loss as a fimction of extrusion temperature than a-LA, presumably due to intermolecular disulfide bond formation. Atomic force microscopy indicates that texturization results in a loss of secondary structure of aroimd 15%, total loss of globular structure at 78 °C, and conversion to a random coil at 100 °C (Qi and Onwulata, 2011). Moisture has a small effect on whey protein texturization, whereas temperature has the largest effect. Extrusion at or above 75 °C leads to a uniform densely packed polymeric product with no secondary structural elements (mostly a-helix) remaining (Qi and Onwulata, 2011). 

On a laboratory scale, the most commonly used E. coli strains are BL21 (protease deficient) and K12 (seems to show enhanced disulfide bond formation) and some optimized derivatives [13] for example, C41 and C43 for membrane proteins expression, Rosetta strains and BL21 CodonPlus strains with overexpressed tRNAs to deal with rare E. coli codons, or JM 83 for proteins secreted into the periplasm. Strains and plasmids are commercially available and were reviewed in several papers [13,26],... 

Braakman, I., Helenius, J., and Helenius, A. (1992). Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J. 11, 1717—1722. 

Even though the short oligosaccharide segment made of seven units may not be considered as a true polymer chain, the chemistry devised by them should be easily applicable to longer saccharidic chains. Liu and Zhang used the Michael reaction for coupling a dextran (Dex) with an amino-functionalized terminal and acrylolyl end-capped PCL [146]. Sun et al. also synthesized Dex-b-PCL by disulfide bond formation [147]. 

The 4,4 -dipyridyl disulfide can be used in aqueous solutions, but it has been found that modification of proteins with this reagent yields rapid disulfide bond formation. Only when 2-iminothiolane is used in tandem with 4,4 -dipyridyl disulfide can 4-dithiopyridyl groups be introduced into proteins (King et al., 1978) (Section 4.1, this chapter). This is due to disulfide interchange reactions predominating without the addition of 2-iminothiolane. 

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

King, P., Li, Y., and Kochoumian, L. (1978) Preparation of protein conjugates via intermolecular disulfide bond formation. Biochemistry 17, 1499. 

Disulfide bond formation Helps stabilize conformation of some proteins... 

The initial approach to recombinant insulin production taken entailed inserting the nucleotide sequence coding for the insulin A- and B-chains into two different E. coli cells (both strain K12). These cells were then cultured separately in large-scale fermentation vessels, with subsequent chromatographic purification of the insulin chains produced. The A- and B-chains are then incubated together under appropriate oxidizing conditions in order to promote interchain disulfide bond formation, forming human insulin crb ... 

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