Intermediates, in protein folding

Creighton, T.E., Darby, N.J., Kemmink, J. (1996) The roles of partly folded intermediates in protein folding. FASEB J. 10, 110-118. 

Morris, G. E (1989) Monoclonal antibody studies of creatine kinase The ART epitope evidence for an intermediate in protein folding Biochem J 257,461-469. 

It is very difficult to obtain experimental evidence for intermediates in protein folding by performing simple rate measurements and one must resort to special flow, relaxation, or trapping techniques to detect intermediates directly. 

Fig. 8.3. Schematic representation of the method using disulfide bonds to trap intermediates in protein folding (courtesy of Creighton, 1978c). A simplified folding pathway is represented. In the quenching reaction each thiol is blocked with a moiety X. The kinetics of the appearance of each species is represented at the bottom.

DETECTION OF INTERMEDIATES IN PROTEIN FOLDING BY DIFFERENTIAL LABELING... 

An exhaustive review of all approaches allowing detection of intermediates in protein folding is not given here. Detection of intermediates during protein... 

The various immunochemical approaches of protein folding indicate the potentiality of the method to (1) study dynamic conformational equilibrium, (2) measure the probability of fragments to reach the conformation they have in the native protein, and (3) detect intermediates in protein folding or to analyze trapped intermediates. The advantages and disadvantages of the... 

Different methods have been used to detect and to characterize intermediates in protein folding,... 

Figure 6.2 The molten globule state is an important intermediate in the folding pathway when a polypeptide chain converts from an unfolded to a folded state. The molten globule has most of the secondary structure of the native state but it is less compact and the proper packing interactions in the interior of the protein have not been formed.

The studies of RNase Aand cytochrome c (Qi etal., 1998 Sosnick etal., 1997) show that caution is required in interpreting burst phenomena in protein folding. However, they do not require a reinterpretation of the cases in which the molten globule character of the burst-intermediate has been established (Arai and Kuwajima, 2000 Chamberlain and Marqusee, 2000). 

Fink, A. L. (1995). Compact intermediate states in protein folding. Annu. Rev. Biophys. Biomol. Struct. 24, 495-522. 

Kim, P. S. and Baldwin, R. L. 1982. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. In Annual Review of Biochemistry, Vol. 51. E.S. Snell, P. D. Boyer, A. Meister and C.C. Richardson (Editors). Annual Reviews, Palo Alto, Calif., pp. 459-489. 

Navea, S., de Juan, A., and Tauler, R., Detection and resolution of intermediate species in protein folding processes using fluorescence and circular dichroism spectroscopies and multivariate curve resolution, Anal. Chem., 64, 6031-6039, 2002. 

Murphy KP, Bhakuni V, Xie D, Freire E (1992) Molecular basis of cooperativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates, J Mol Biol, 227 293-306... 

Freire, E. (1995). Thermodynamics of Partly Folded Intermediates in Proteins. Ann. Rev. of Biophys. and Biomolec. Struct. 24, 141-165. 

---