Protein unfolding kinetics

It should be noted that the unfolding kinetics can sometimes involve quite complex unfolding schemes of different substates in equilibrium with the native state. Staphylococcal nuclease is an example of such behavior, known to unfold with three different substates that exhibit an equilibrium that does not appear to shift with temperature.49 Irreversible aggregation processes of proteins have been known to involve first- or second-order reactions.132141 The mechanism of recombinant human interferon-y aggregation is an example where thermodynamic and kinetic aspects of the reaction provided a powerful tool for understanding the pathway of instability and permitted a rationale for screening excipients that inhibited the process.141... 

Biochemical processes such as protein unfolding/refold-ing and supramolecular assembly/disassembly take place on a time scale of seconds to minutes after readjusting the temperature of a system. Most commercially available glass-jacketed cuvettes are not suitable for temperature jumps on this time scale, as a result of the slow kinetics of heat transfer across substances with characteristically high dielectric constants, and their use can convolute the time scale of the temperature change onto the time scale... 

This kind of observation has led to the idea that functional RNA molecules tend to fold an order of magnitude more slowly than proteins, as kinetic traps may form on the folding pathway which then take time to unfold again so that the molecule can finally reach its functional folded state. However, the lack of time-resolved RNA folding measurements to date on a range of different functional RNAs means that this idea has not yet been substantiated in depth. 

Although the rate of mutarotation of cold dilute gelatin is independent of protein concentration, kinetic analysis reveals an exponential dependence of d[a]/dl on the concentration of chain elements in the unfolded form. Thus van t Hoff plots of log (d a]/dt) versus log ([ ] —[ ]<), derived from the general equation ... 

Ion mobility spectrometry (IMS), in which ions are separated on the basis of differences in the cross-sectional areas, can be used to determine the conformation and folding—unfolding kinetics of proteins.151,152 The basic idea behind these measurements is that because of their distinct shapes, different conformers will travel at... 

Here, and k2 are the unfolding rate constants for the unfolding transition from N to I and from I to respectively. The unfolding kinetics can be quantitatively analyzed from the scanned intensities of the Coomassie blue stained tailspike bands on SDS gels. Figure 1 depicts results from a typical thermal unfolding experiment for wild type tailspike protein, which was performed in Tris buffer 8) and 2% SDS at 65 C. Kinetic analysis yields two rate constants 1.1 x 10 s and 4.0 x 10 s for the conversion from N to I and from I to A/, respectively. 

Solutes are one of the major components of foods, and they have significant effects on their adsorption at fluid interfaces. In addition, the study of the effects of ethanol and/or sucrose on protein adsorption at fluid interfaces is of practical importance in the manufacture of food dispersions. The presence of ethanol in the bulk phase apparently introduces an energy barrier for the protein diffusion towards the interface. This could be attributable to competition with previously adsorbed ethanol molecules for the penetration of the protein into the interface. However, if ethanol causes denaturation and/or aggregation of the protein in the bulk phase, the diffusion of the protein towards the interface could be diminished. The causes of the higher rate of protein diffusion from aqueous solutions of sucrose, in comparison with that observed for water, must be different in aqueous ethanol solutions. Since protein molecules are preferentially hydrated in the presence of sucrose, it is possible that sucrose limits protein unfolding in the bulk phase and reduces protein-protein interactions in the bulk phase and at the interface. Both of these phenomena may increase the rate of protein diffusion towards the interface. Clearly, the kinetics of adsorption of proteins at interfaces are highly complex, especially in the presence of typical food solutes such as ethanol and sucrose in the aqueous phase. 

Scheme 1. Kinetic model for the coupling between protein unfolding and prolyl isomerization.

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