Renaturation models

COMMENTS ON THE VALIDITY OF THE IN VITRO DENATURATION-RENATURATION MODEL... 

For several proteins the conditions of reversibility are very restricted. Conditions such as pH, ionic strength, temperature, and even residual concentration of denaturant must be carefully determined in the study of the refolding of a protein. The narrower the conditions of renaturation, the fewer are the probable possible pathways to refold a protein. Nevertheless, the presence of ribosome is not a necessary requirement to refold a protein and the in vitro denaturation-renaturation model may be considered very significant, at least for soluble globular proteins. 

Fig. 15. Suggested general kinetic model for protein adsorption in the absence of any covalent bond formation or disruption. Any protein desorbed in a denatured state is assumed to rapidly renature in solution. If the surface is heterogeneous, then two or more such scenarios can be formulated, with appropriate account of the area fractions of each type of surface present...

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. 

The use of . coli as an expression system is limited to those proteins where post-transla-tional modification such as the glycosylation or galactosylation of antibody fragments is not required. Inclusion body formation can be another disadvantage that occurs with this prokaryotic model, as these insoluble protein aggregates demand laborious and cost-intensive in vitro refolding (denatura-tion and renaturation) and purification steps. 

The determination of binding and conformational changes leaves the question of the detailed structure of complexes unanswered. At present there is no absolute method for structure determination of protein-surfactant complexes apart from x-ray diffraction, which has only been applied to lysozyme with three bound SDS molecules [49]. X-ray diffraction requires a crystal, so in the case of lysozyme cross-linked triclinic crystals of the protein were soaked in 1.1 M SDS and then transferred to water or a lower concentration (0.35 M) of SDS to allow the protein to refold. It was necessary to use cross-linked crystals to prevent them dissolving when exposed to a high SDS concentration. The resulting denatured-renatured crystals were found to have three SDS molecules within a structure that was similar but not identical to that of native lysosyme. Neutron scattering has been applied in a few cases (see Sec. IX), but this is a model-dependent technique. 

Figure 4.6.3 Repeating unit of scleroglucan and a model of a triple helix in water and of a toroid formed after denaturation in DMSO and renaturation in water.

The gelatin-type of triple heUx may be considered a type of crystal indeed, the denaturing and renaturing of these gels is a first-order transition. However, it has been modeled as a nucleated, one-dimensional crystallization, as opposed to the ordinary three-dimensional crystallization of bulk materials. 

Figure 2.8. Model proposed by Holzwarth and Prestridge (1977) to explain their electron microscopy observations on xanthan. The section of native xanthan is thought to denature into shorter strands because of breaks in one or other chain of the double helix. The renaturing process is a reverse procedure but imperfections arise during the reassembly, causing small hockles , as shown in the renatured molecule.

Special attention has been given to the distribution of unique and repeated sequences along the chromosome and to the construction of polytene chromosomes. Hypotheses on chromomere construction in Drosophila have been based on data of DNA renaturation kinetics (Laird and McCarthy, 1969), formation of rings from DNA fragments (Lee and Thomas, 1973 Thomas et al., 1973), and electron microscopic analysis of repeated sequences (Wu et al., 1972). The models of chromomere construction are presented in Fig. 104 (Laird, 1973). 

The phenomenon of denaturation and renaturation can ideally be treated using a two-state model involving the transition between ordered double helices and disordered or random coils. When the transition monitored by A260, fot... 

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