The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway

Abstract

An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (1 and 2) defining two competing pathways in the folding energy landscape: 1 3 2 and 2 3 1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate. circular permutation ͉ energy landscape ͉ protein dynamics ͉ transition state I n contrast to the reaction pathways of small molecules, the process of protein folding is typically malleable and responds to sequence divergence (1), circular permutation (2) and changes of the experimental conditions (3). In several cases, this malleability follows specific and predictable patterns that can be used to uncover the topology of the folding-energy landscapes and the rules that define them (4, 5). An extensively characterized system in this respect is the ribosomal protein S6 (1RIS) (6). Upon circular permutation of the S6 sequence, the interactions of the folding transition-state ensemble show pronounced redistributions that follow simple entropic rules: the -values increase with decreased sequence separation between the targeted contacts (2, 7, 8). However, the data reveal also a more global pattern. The -value changes describe redistributions between two competing folding nuclei. Both of these nuclei seem comprised by a basic two-strand-helix motif but with different locations in the S6 structure. One corresponds to the tertiary condensation of ␤1, ␣1, and ␤3 (1) and the other to the condensation of ␤1, ␣2, and ␤4 (2) at the original N and C termini (Fig. 1). Because of the dual initiation points, the folding process of S6 can proceed along either of two parallel pathways. One is running in the order 1 3 2 and the other 2 3 1 (Fig. 1). Hence, permutations that redistribute the flux of molecules from one pathway to the other will also reverse the sequence of folding events. The phenomenon is nicely exemplified by the folding behavior of S6 wt , which is biased mainly toward the 1 3 2 pathway, and the permutant P 54-55 , which uses predominantly the reversed 2 3 1 pathway. This modular architecture of multiple, ...

Folding without charges

“…S2). The results yield a linear correlation of r ¼ 0.95, which closely resembles that between wild-type S6 þ17−17 and the structurally ordered permutant S6 54−55 (23,24) (Fig. S2), and supports the conclusion that K and R removal does not significantly alter the S6 structure.…”

“…Folding of S6 is a two-state process with two competing pathways, the bias between which can be altered by circular permutation (24,25) (Fig. S3).…”

Prediction of Native-State Hydrogen Exchange from Perfectly Funneled Energy Landscapes

“…For example, compare the free energy difference between the native and unfolded state basins of ubiquitin at the experimental temperature (red curve in Figure 5A), with the predicted ln Pf pattern of the protein at the same temperature (magenta curve in Figure 5B). This result, that all residues become open in the unfolded state, sets an upper limit for the free energy difference between the open and closed states 40 .…”

Trimming Down a Protein Structure to Its Bare Foldons

“…protection factors as measured by hydrogen-exchange NMR (9); in contrast to the 1 foldon (Fig. 1), which exchanges by global unfolding (EX1), ␣2 and ␤4 exchange by local motions in the native free-energy basin (EX2), suggesting that these secondary structure elements can be removed with maintained folding barrier.…”