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All roads lead to Rome? The multiple pathways of protein folding

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All roads lead to Rome? The multiple pathways of protein folding Abstract Recent studies have found that protein folding reactions often proceed through two or more kinetically distinct pathways. In at least some cases, the observed folding intermediates act as kinetic traps, slowing the rate at which folding is completed. These findings have important implications for understanding how proteins fold in vitro and in vivo. Introduction Efforts to understand how proteins adopt their native three-dimensional structure have been strongly influenced by two observations. The first, due to Anfinsen and coworkers [1], is that proteins can fold spontaneously on a physiological timescale. The second, due to Levinthal [2], is that there are vastly too many conformations open to a protein for all of the possible states to be sampled. These observations seem, on the face of it, to be mutually exclusive. To resolve this paradox, it was proposed that proteins fold via specific pathways. Much of the experimental work on protein folding has concentrated on identifying folding intermediates and, in analogy to simple chemical reactions, ordering these intermediates into a discrete folding pathway [3]. Over twenty years of research has produced a wealth of data on the energetic and structural nature of protein-folding intermediates [4] [5]. It is now clear that during the folding of most proteins, specific structural intermediates, often containing significant stable secondary structure, are formed rapidly. The kinetic role of the observed intermediates, however, is less well established. An implicit assumption in many folding studies is that the observed intermediates are critical in restricting the conformational space sampled by the polypeptide, thus allowing it to fold rapidly. In the simplest cases, the intermediates would form a direct linear pathway (Fig. 1a). Alternatively, the observed intermediates might assist folding but could be ordered in two or more distinct competing pathways (Fig. 1b). Recently, however, it has been found that the folding of several proteins can occur extremely rapidly (~10-50 msec) ...read more.


Thus, the different folding channels arise too rapidly to be due to proline isomerization. In addition, replacement of a cysteine residue, not located near the prolines in native DHFR, causes collapse of the four folding phases to two [13]. Since DHFR contains no disulfide bonds, replacement of this cysteine residue cannot be affecting the formation of different disulfide species. Finally, folding of hen egg white lysozyme has also been observed to involve parallel pathways and distinct folding domains [14]. The native structure of lysozyme consists of two lobes separated by a cleft. The first lobe is predominantly ?-helical and the second predominantly ?-sheet. The folding of lysozyme has been studied extensively by a number of methods, including the use of pulsed-amide hydrogen-deuterium exchange. Amide hydrogens are readily exchanged with the solvent in unfolded proteins, but this exchange is often strongly inhibited when a protein folds. By measuring the rate of amide-hydrogen exchange as a function of folding time, it is possible, therefore, to monitor the formation of structure during the folding reaction. For the lysozyme studies, the rate of hydrogen exchange was examined by a combination of two-dimensional NMR, which measures the average rate at which individual amide protons exchange, and electrospray ionizing mass spectrometry, which distinguishes between different folding populations. At 20 msec, two major folding phases were detected: one in which the -domain had achieved a high degree of protection while the ?-domain contained no persistent structure, and a second in which no stable structure is observed in either the ?- or ?-domains. In addition, a third, less well populated pathway is observed in which both the ?-domain and the ?-domain are significantly protected. This heterogeneity is not due to residual structure in the unfolded state, as the ratio of the different populations does not depend on the denaturant used to unfold the protein [15]. In some cases, there may be only one major pathway In contrast to the above results, recent studies from Fersht et al. ...read more.


It has also been found that, as well as preventing off-pathway aggregation reactions, under some circumstances GroEL can increase the rate of folding by rescuing stable but unproductive intermediates in the folding of RuBisCO (ribulose 1,5-bisphosphate carboxylase-oxygenase) [25] and malate dehydrogenase [26]. The above considerations suggest that both Hsp70 and chaperonins might function in part by binding to kinetically trapped, slowly folding, or even mildly aggregated folding intermediates [8] (Fig. 3). This binding could facilitate the rearrangement of intermediates with incorrect proline isomers as well as the unfolding of kinetically trapped species. Finally, binding and/or hydrolysis of ATP would drive release of the unfolded protein. Folding could then be completed either in the bulk solution or perhaps while the protein remains associated with the chaperone. In this way, molecular chaperones could increase the fraction of the molecules that fold through rapid folding phases. This channeling of folding through inherently fast phases could lead to the observed rate enhancements sometimes seen in chaperone-assisted folding. Fig. 3. Hypothetical model for one mechanism by which molecular chaperones could increase the efficiency of folding. U designates the fully unfolded state present under strongly denaturing conditions. Ip. designates a productive protein-folding intermediate. Ik designates a kinetically trapped or perhaps mildly aggregated intermediate. Conclusions Folding of a protein is a vastly complicated reaction involving thousands of independent degrees of freedom. It is not surprising, therefore, that many folding reactions involve complex kinetics and multiple phases. Although the rapid rate of folding requires that the conformational space explored by a folding protein be highly restricted, it does not follow that the kinetic intermediates observed are involved in guiding a protein to its native states, nor is there any requirement that a protein fold by discrete pathways [27] [28]. Much work needs to be done to establish the kinetic role of observed intermediates. These efforts should be aided by new statistical mechanical models [29] [30] being developed which may provide a firm theoretical framework for understanding complex folding reactions. Acknowledgements I thank C.R. Matthews and S.E. Radford for helpful discussions. Due to space limitations, not all relevant references could be cited. ...read more.

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