Renaturation residues

A polypeptide consisting of residues 191-345 of GroEL that, when immobilized on agarose, acts as a very efficient chaperone with proteins that resist renaturation by conventional refolding methods. Immobilized minichap-... 

Peptides consisting of residues from GroEL immobilized on agarose have proved effective minichaperones (Altamirano ef al., 1997). The procedure used both column chromatography and batch-wise methods to renature an insoluble protein from an inclusion body, refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Eragments were immobilized by two methods Ni-NTA resin and CNBr-activated Sepharose 4B. 

Reactivation of alkaline phosphatase is of considerable practical significance since regulatory tests for pasteurization assume the absence of phosphatase activity. An official AOAC method used to distinguish between renatured and residual native alkaline phosphatase is based on the increase in phosphatase activity resulting from addition of Mg2 + the activity of renatured alkaline phosphatase is increased about 14-fold but that of the native enzyme is increased only two-fold. 

Urea is used to denature ribonuclease, and mercaptoethanol (HOCH2CH2SH) to reduce and thus cleave the disulfide bonds to yield eight Cys residues. Renaturation involves reestablishment of the correct disulfide cross-links. 

Kinetic analysis indicates that renaturation is a two-step process. In the slow step effective contact is made between two complementary regions of DNA originated from separate strands. This rate-limiting step called nucleation is a function of the concentration of complementary strands. Nucleation is followed by a relatively rapid zippering up of adjoining base residues into a duplex structure. The steps involved in denaturation and renaturation are depicted in figure 25.14. 

P40 contains 344 amino acid residues. For ES-MS analysis, since the detergent used for renaturation and purification of P40 is incompatible with subsequent spectrometric analyses, it was eliminated. The protein was precipitated by addition of cold ethanol. Following the removal of detergent, mass measurement of the purified P40 was performed by ES-MS after solubilization in formic acid. The measured mass, 37.061 +/- 2 Da, was in accordance with the theoretical mass (37.059 Da) deduced from the DNA sequence (Fig. 8). 

The properties of 3-D-fructofuranosidase (invertase) of Saccharomyces cerevisiae, and of the modified enzyme after removal of about 90% of the carbohydrate residues, have been found to differ considerably, not only in stability but also in their capacity to be renatured following inactivation by guanidinium chloride. The covalently bound carbohydrate of yeast 3-D-fructo-... 

The renaturation process of globular proteins takes place within 10 and 10" seconds. Internal molecular rotations are known to be in the order of 10 seconds Taking into account only three energetically favorable conformational states for each amino acid, a relatively small protein of 150 residues can potentially adopt more than 10 conformations. A comparison of these figures illustrates in an impressive manner that the time available to a protein for the folding process is by far not sufficient to find the energetically most favorable conformation by a random search mechanism. 

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