Cysteine-containing proteins, folding rate

Proteins that contain disulfide bonds often fold slowly in vitro because the oxidation and coixect pairing of the cysteine residues becomes the rate-limiting step and the bonds formed ai e not always the coiiect ones. Many proteins, especially those that are secreted by eu-kaiyotes, aie stabilized by disulfide bonds. Examples of such proteins include those used for medical or biotechnological puiposes, such as interleukins, IFNs, antibodies and then fragments, insulin, TGF, and many toxins and proteases. Expression of recombinant proteins as inclusion bodies in bacteria can be a very efficient way to produce cloned proteins, as long as the inclusion body protein can be successfully refolded. 

With the exception of disulfide bonds, all post-translational modifications must be catalyzed by cellular enzymes. The formation of disulfide bonds can occur at appreciable rates in the absence of enzymes and involves two steps (i) the relatively rapid pairing of cysteines to form S-S bonds that do not correspond to those in the native structure and (ii) disulfide rearrangement (20), The isomerization of disulfide bonds to form the correct cysteine pairs that are present in the native protein is slow and represents an important rate limiting step in folding. For this reason the in-vitro refolding of polypeptides containing several cysteines is usually very slow and inefficient. In eucaryotic cells the formation of the correct disulfide bonds is accelerated by the enzyme protein disulfide isomerase or PDI (38,39), PDI is located... 

E. coli LipB contains three cysteinyl residues, and treatment of the protein with 5,5 -dithiohis-(2-nitrohenzoic acid) as a function of time showed that one cysteine is modified with a rate constant that is 50-fold greater than that of the other two. " An independent study by the Cronan laboratory showed that E. coli LipB catalyzes its reaction through a ping-pong mechanism, in which the octanoyl group from octanoyl—ACP is first transferred to Cysl69 of LipB before it is transferred to the appropriate LCP. ... 

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