Protein folding denaturant concentration

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

Incubation in solutions of low denaturant concentration can sometimes cause inactivation. Formation of an incorrectly folded form of human carbonic anhydrase in 1.7M GuHCl which subsequently aggregates has been documented (77). Other proteins have also been observed to improperly refold at relatively low concentrations of denaturant These proteins include 6-galactosidase, tryptophanase, and elastase (6). 

This renaturation procedure may find wider application to the commercial-scale renaturation of other recombinant proteins from bacterial inclusion bodies. The only proviso appears to be the existence of one or possibly more partially folded states at intermediate denaturant concentrations. Structural homologs of BST such as porcine somatotropin (16) and bovine prolactin are candidates for this approach to refolding. We are currently developing a general model for the prediction of optimum denaturant concentration for protein refolding via a consideration of the tradeoffs between aggregation and unfolding phenomena. 

What guides proteins to their native folded state The answer to this question Initially came from In vitro studies on protein refolding. Thermal energy from heat, extremes of pH that alter the charges on amino acid side chains, and chemicals such as urea or guanidine hydrochloride at concentrations of 6-8 M can disrupt the weak noncovalent Interactions that stabilize the native conformation of a protein. The denaturation resulting from such treatment causes a protein to lose both Its native conformation and Its biological activity. 

The volume of native Tendamistat is increased by 41.4 + 2.0 cm mol" compared with the denaturated form at pH 2.0 and 35 °C (Fig. 11.2b). This value is virtually independent of the denaturant concentration and it is similar to reaction volumes (AV°) for folding of many other small single-domain proteins [6]. Contributions to AV° may arise from packing deficiencies in the native state as well as from different solvent interactions in the native and in the unfolded protein. Although the reaction volume is virtually independent of the denaturant concentration, the activation volumes for refolding and unfolding both greatly increase with... 

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