Woes of proline: A cautionary kinetic tale

This tale pushes hot buttons in protein science, including the causes of slow folding, trapped but obligatory intermediates, and alternative native states. As with all good tales, there is a moral, one that is especially pertinent to newly minted graduate students applying their course-work to research. Let’s start with proline. Unlike the other 19 ribosomally encoded amino acids, proline’s side chain bites back into its α-nitrogen. Most peptide bonds in proteins favor the trans configuration because it puts the α-carbons far away from each other. In X-pro, where X is any of the other 18 side-chain-containing amino acids, this contortion causes a steric clash between the α-carbon on X and both the α-and the N-bound δ-carbons on pro, and the usually 500:1 ratio of trans to cis peptide bonds decreases to 4:1. Furthermore, cis–trans isomerization of X-pro bonds can take minutes. These features—two populated states and slow interconversion—can complicate folding kinetics when fast dilution of a urea-denatured protein is used to induce folding. The complication is the observation of fast and slow phases. These phases are often explained based on the isomers of X-pro bonds in the denatured state. Molecules with the native X-pro isomer fold quickly. Those with the nonnative isomer must wait to isomerize before folding; their folding rate is limited by isomerization. As shown in a careful study in this issue (Chen and Clark 2006), however, slow does not have to mean pro. Chen and Clark (2003, 2006) focus on the 10-kDa caspase recruitment domain. Its refolding occurs in four phases: a phase that is too fast to measure (the burst) and phases with half-lives of 22 msec, 0.7 sec, and 60 sec. The simplest explanation is that the burst phase represents the population of unfolded molecules with the native X-pro isomer and the 60-sec phase represents the population that must undergo rate-limiting proline isomerization. These suspicions were reinforced by the observation that the slow reaction is unaffected by urea concentration because increasing urea concentration should not affect X-pro isomerization. “Double-jump” experiments poured cold water on the idea of rate-limiting X-pro isomerization (Chen and Clark 2003). In these experiments, the native state is mixed with denaturing concentrations of urea, and the sample held there for various times before being returned to native conditions (Brandts et al. 1975). At short times, X-pro bonds remain in their native isomeric state, and refolding is fast. The longer the wait, the greater the isomerization, and the larger the amount of the slow phase. Chen and Clark (2003) observed something strange: Instead of seeing the burst phase decrease with increasing delay time, they had to wait to see the burst phase. This result is inconsistent with a simple set of parallel pathways that differ by the proportion of X-pro isomer. Instead, these and other data led to the proposal that instead of rate-liming X-pro isomerization, folding occurs sequentially via at least one kinetically trapped, or misfolded, obligatory intermediate. The idea that trapped/misfolded intermediates are obligatory (i.e., on a direct path to the native state) is controversial. To determine if these intermediates involve proline isomerization, Chen and Clark (2006) studied all combinations of proline-to-alanine variants. Double-jump experiments showed that all the variants still had slow phases, ruling out rate-limiting proline isomerization. Like the wild-type protein, the burst phase is not observed until the protein sits under denaturing conditions, but the slow phase is observed at short delay times. These observations argue against trapped obligatory intermediates and for parallel pathways with slowly interconverting species (Fig. 1​1).). Complicating matters further, the proline variants affect the distribution of these species. Yet we are still not at the end of the tale. Figure 1. A mechanism for folding the caspase recruitment domain. Ix and Iy represent at least one intermediate. The dashed lines represent potential interconversion of the intermediates. Anfinsen’s (1973) thermodynamic hypothesis states that the native state of a globular protein is at the free energy minimum, which means there is but one native state at equilibrium. We expect this to be true at all times during folding. The last set of experiments described in the current paper involves “interrupted folding” (Schmid 1983), in which the urea-denatured protein is subjected to native conditions for various times and then returned to denaturing conditions. The investigators found that the protein unfolds like the native protein only after it had been held at folding conditions for 100 sec. This observation means that fastest folding phases do not lead directly to the native state. Instead, there is more than one “native state” at short folding times. These alternative native states require hundreds of seconds to find the equilibrium native state. In summary, we have slow folding that is not caused by proline, a pathway that started out looking sequential with obligatory intermediates but is probably parallel (Fig. 1​1),), and a protein with more than one native state. The moral is that only the most simple material is taught in class, and proteins missed those lectures.