Modulation of frustration in folding by sequence permutation

Abstract

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.

Keywords: CF-SAXS; CheY permutants; Gō models; protein-folding intermediates.

Fig. 1.

(A) Topology diagram of CheY. The N-terminal folding subdomain is highlighted in yellow, and the C-terminal folding subdomain is highlighted in blue. The effects of each permutation on the continuity of cluster 1 (blue), and cluster 2 (red) are shown. (B) Clusters of ILV residues are superimposed on the crystal structure of CheY (Protein Data Bank ID code: 3CHY). Cluster 1 (blue) has a lower CO and resides on the α2/α3/α4 side of the central β-sheet. The larger cluster 2 (red) contains high-CO contacts and resides on the α1/α5 side of the β-sheet. (C) The folding mechanism of WT CheY. The major pathway is highlighted in red. The U_c →N_c step, involving the on-pathway intermediate, is designated by the triple dots.

Fig. 2.

Analysis of N and I_BP stability. Filled symbols display the urea melts derived from the ellipticity at 222 nm for CheY* and each of the permuted variants; the denaturant-induced unfolding reactions are fully reversible. The open symbols display the urea dependence of the ellipticity at 222 nm after 5 ms of refolding. With the exception of Cpβ4, the solid and dashed lines show the fits of these data to two-state equilibrium models. The data for Cpβ4 are fit to a three-state model.

Fig. 3.

Dimensional analysis of CheY* and Cpβ4 during folding by SAXS and simulations. The radius of gyration for CheY* (black) and Cpβ4 (red) from CF-SAXS (A) and the average R_g from Gō-model simulations (CheY*: n = 46; Cpβ4: n = 32) in which the intermediate was observed (B) as a function of folding time. Statistical analysis of the simulations finds the intermediate to be highly populated within the average time values of the first and last occurrences (green box; see Table S2 for details).The unweighted R_g values of I_ON and I_OFF species from simulations are shown as dotted lines. Arrows indicate the R_g values and their estimated uncertainties under equilibrium conditions for the folded and the unfolded states (A). Ninety-three points were collected within the mixer channel from 142–2,400 μs and averaged over 20 scans. After low-quality data points were removed, the remaining data were binned into two parts, 142–959 μs and 1,055–2,400 μs. CheY* Rg = 25.3 ± 2.2 Å (n = 11, 142–791 μs) and 22.6 ± 2.0 Å (n = 15 1,223–1,944 μs). Cpβ4 Rg = 18.0 ± 0.7 Å (n = 21, 142–959 μs) and 17.8 ± 0.7 Å (n = 33 1,055–2,304 μs).

Fig. 4.

Frustration observed in Gō-model simulations. (A–C) Ensemble averaged fractional contacts of the N-terminal subdomain (red), C-terminal subdomain (blue), and subdomain interface (green) are plotted as a function of fractional total native contacts for CheY* (A), Cpβ4 (B), and Cpβ3 (C). (D–F) The interfacial region is dissected in D–F where β3–β4 contacts are shown in magenta, α2–α3 contacts are shown in black, and α5–C-terminal contacts are shown in green. The C-terminal subdomain is dissected into fragments of β4–β5 contacts (gold) and α3–α4 contacts (blue) for CheY* (D), Cpβ4 (E), and Cpβ3 (F).

Fig. 5.

Structures of intermediates and the simplified folding free-energy surfaces. The sequence of events in folding is indicated by the arrows. The proline isomerization step, occurring between the cis and trans I_BP species, is not shown. Structured components of each species as determined by Gō-model simulations. Elements in gray are not yet formed; colored elements [A: black, CheY*; B: red, Cpβ4; C: blue, Cpβ3] are significantly structured; elements implicated in topological frustration are orange. (D) Reaction coordinate diagrams for CheY* (black), Cpβ3 (blue), and Cpβ4 (red). The barrier heights were estimated using the Kramer’s formalism with a prefactor of 1 μs, and m-values were calculated from equilibrium and kinetic experiments, when available. Each permutant would have a unique unfolded ensemble, but the free energies have been aligned for direct comparison.