Unfolding pathway, kinetic

Series first-order processes are especially important in understanding metabolic pathway kinetics. Likewise, one should recognize that unassisted protein folding " is unlikely to be a single-step reaction (say U N), where U represents unfolded protein, and N is the native conformation. At a minimum, realistic schemes for protein folding are apt to occur as a series of isomerizations ... 

In any case, the difference between the in vivo folding pathway and the in vitro thermal unfolding pathway indicates the importance of kinetic aspects in controlling the folding and unfolding of this protein. 

Bamase, like CI2, has been the subject of extensive studies, using the protein engineering method combined with kinetic and equilibrium experiments, to investigate its fold-ing/unfolding pathway." Molecular dynamics simulations of thermal denaturation of the wild-type and two mutant proteins (Tyr 17 —> Gly and He 88 —> Val) have been performed to investigate the effect of mutations on the unfolding pathway and transition state." ... 

Given that a sequence folds to a known native stmcture, what are the mechanisms in the transition from the unfolded confonnation to the folded state This is a kinetics problem, the solution of which requires elucidation of the pathways and transition states in the folding process. 

For these sequences the value of Gj, is less than a certain small value g. For such sequences the folding occurs directly from the ensemble of unfolded states to the NBA. The free energy surface is dominated by the NBA (or a funnel) and the volume associated with NBA is very large. The partition factor <6 is near unify so that these sequences reach the native state by two-state kinetics. The amplitudes in (C2.5.7) are nearly zero. There are no intennediates in the pathways from the denatured state to the native state. Fast folders reach the native state by a nucleation-collapse mechanism which means that once a certain number of contacts (folding nuclei) are fonned then the native state is reached very rapidly [25, 26]. The time scale for reaching the native state for fast folders (which are nonnally associated with those sequences for which topological fmstration is minimal) is found to be... 

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

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... 

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. 

The biological activity of proteins generally depends on a unique three-dimensional conformation, which in turn is inherently linked to its primary sequence. Protein folding, the conversion of the translated polypeptide chain into the native state of a protein, is the critical link between gene sequence and three-dimensional structure. Mechanistically, folding is believed to proceed through a predetermined and ordered pathway, either via kinetic intermediates or by direct transition from the unfolded to the native state [99]. In both cases, local and non-local interactions alike stabilize transient structures along the pathway and funnel the intermediates towards the native state. 

For a mechanistic understanding of protein folding, it is necessary to determine the sequence of intermediate states between the fully unfolded and fully folded forms of a polypeptide chain (i.e. the folding pathway) and to describe the major intermediates in structural and kinetic terms. One of the... 

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