Transiently populated intermediate functions as a branching point of the FF domain folding pathway

Abstract

Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.

Fig. 1.

(A) The folding pathway of the FF domain consists of a fast microsecond transition from U to I and a slower millisecond conversion from I to N. Structural ensembles of I [Protein Data Bank (PDB) ID code 2KZG] and N (PDB ID code 1UZC) were calculated as described elsewhere (13, 23). (B) Selected region of the HSQC spectrum of the truncated variant, FF_1–60, a mimic of the folding intermediate of the full-length domain (11.7 T, 25 °C). Two sets of signals are observed, derived from two separate conformations of the protein, denoted as M and D. (C) Backbone , ¹⁵N, , , and chemical shift differences between I and N states of the full-length FF domain (FF_1–71) obtained from RD NMR data (13) (), plotted vs. chemical differences between FF_1–60 (M form) and the native state FF_1–71 (). ϖ_std,i is a nucleus- and residue-specific normalization value that corresponds to the range of shift values (1 SD) that are observed in a database of protein chemical shifts (www.bmrb.wisc.edu) for the nucleus/residue in question. A similar correlation for the D state of FF_1–60 is shown (Inset).

Fig. 2.

(A) Selected region of a magnetization exchange spectrum (24, 25) of 0.34 mM FF_1–60 recorded with a mixing time T = 0.34 s (11.7 T, 25 °C), showing two sets of autopeaks (MM and DD) connected by exchange cross-peaks (MD and DM) for Ala34. (B) Mixing time, T, dependencies of autopeak (circles) and exchange cross-peak (boxes) volumes for Ala34 from magnetization exchange experiments. The solid lines are generated from a least-squares fit of the exchange data for Ala34 to Eq. S2 in SI Materials and Methods, resulting in k_MD = 3.34 ± 0.17 s^-1 and k_DM = 1.97 ± 0.09 s^-1; averaging over five residues with a full set of four non-overlapped auto- and cross-peaks gives k_MD = 3.05 ± 0.95 s^-1 and k_DM = 2.16 ± 0.76 s^-1. (C) Apparent backbone ¹⁵N R₁ and R₂ relaxation rates as a function of residue for correlations derived from M (red circles) and D (green circles) states of FF_1–60 obtained by single-exponential fits of peak volumes in a series of two-dimensional correlation spectra measured in conventional ¹⁵N R₁ and R_1ρ experiments (28, 29). As discussed in the text, the apparent ¹⁵N R₁ and R₂ rates are affected by slow exchange between M and D states; intrinsic relaxation rates (solid lines) are calculated using the procedure described in SI Materials and Methods. Secondary structural elements are indicated at the top of the panel. Helix H2 of state D may extend further than Met42 (denoted by dashed line after residue 42); however, correlations for Ile43 and beyond were missing in spectra of the dimeric form. (D) Cross-peak volumes for Ala34 measured from HSQC spectra of FF_1–60 recorded as a function of protein concentration. The volumes are normalized by total protein concentration.

Fig. 3.

(A) Backbone chemical shift differences between monomeric (M) and dimeric (D) forms of FF_1–60, , as described in the text. Values of are shown for residues Thr8-Met42; the chemical shifts of the first seven residues are indistinguishable in the two forms, while those from the C terminus (residues Ile43-Gln60) are missing in NMR spectra of the dimer. (B) TALOS-plus predicted (33) α-helix probability, P (helix), plotted vs. residue for states M (red) and D (green, residues Thr8-Met42 only). (C) RCI-predicted (34, 35) order parameters, S², for the backbone amide groups of M (red) and D (green).