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) in an all-or-none manner that does not involve detectable folding intermediates (see [6] for discussion). These findings raise the issue of whether the observed intermediates generally assist folding, or whether they are the products of off-pathway reactions, having structures that are irrelevant or even detrimental to the formation of the final native state (Fig. 1c).

Fig. 1. Three schemes for the kinetic role of protein-folding intermediates. (a) A simple sequential pathway in which the observed intermediates are required to assist folding. (b) A set of parallelpathways in which the folding intermediates accelerate folding, but different competing parallel pathways exist. (c) A set of dead-end pathways in which the observed intermediates are kinetically trapped, dead-end species whose structure must be partly, or completely unfolded before folding is completed.

Experimental evidence for multiple folding pathways

One of the first protein-folding pathways to be studied, and still among the best characterized, is that of the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) [7] [8]. Protein-folding intermediates are generally transient in nature, making them difficult to characterize. Proteins like BPTI, in which folding is thermodynamically linked to the formation of a set of native disulfide bonds, provide an exception, however. In such cases, it is possible to reversibly trap the disulfide intermediates present during the folding reaction and identify them. A particularly powerful aspect of these studies is that the trapped intermediates can be purified and allowed to re-enter the folding pathway. Thus, one can evaluate the kinetic role of a given species directly [9]. During the folding of BPTI at neutral pH, two species, each containing a single native disulfide bond, accumulate transiently (Fig. 2a). A second native disulfide bond in both of the well populated single disulfide species is formed rapidly, yielding two intermediates termed N' and N*. In contrast to the well populated single disulfide intermediates, which are in rapid equilibrium, the rate of interconversion between N' and N* is extremely slow. Moreover, the N' intermediate forms native BPTI ~30-fold more rapidly than does N*. Thus the folding of BPTI occurs via two distinct kinetic phases. Interestingly, these two phases are also observed in the presence of the enzyme protein disulfide isomerase, even though the overall rate of folding is increased dramatically [10].

Fig. 2. Examples of folding reactions involving multiple distinct pathways. (a) Schematic representation of the kinetically preferred pathway for the folding of BPTI at pH 7.3, 25 °C [8]. All of the well populated intermediates contain only disulfide bonds found in native BPTI. R denotes the reduced protein. Qualitative descriptions of the relative rates of the intramolecular transitions associated with each step are indicated. The dotted arrows indicate that R is oxidized initially to a broad distribution of one-disulfide intermediates, which then rearrange rapidly to the intermediates containing disulfide bonds 30–51 and 5–55. A fork in the pathways is observed: the 30–51 intermediate yields a native-like species, termed N', which forms native BPTI on the hour timescale whereas the 5–55 species yields a kinetically trapped species, termed N*, that is stable for up to a week in these conditions. (b) Pathway for folding of E. coli DHFR [12]. Four distinct kinetic phases are observed leading to four native-like species. Only one of these species can bind the co-factor NADPH. Kinetic and mutagenesis studies indicate that these phases do not result from proline isomerization (see text and [12] [13]).

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A major source of heterogeneity in the folding reaction of a number of proteins, including ribonuclease A, ribonuclease T1 and staphylococcal nuclease, results from the slow rate of proline isomerization [11]. Prolines are unusual in that the peptide bond preceding these residues is often found in a cis configuration. Even in denatured proteins, the rate of isomerization of the cis–trans peptide bond is often slow compared to the rate of folding. As a consequence, those unfolded molecules that contain incorrect proline isomers frequently fold at a relatively slow rate. A number of tests have been developed to determine whether ...

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