Characterization of a transient unfolding intermediate in a core mutant of γS-crystallin

Abstract

In many age-related and neurological diseases, formerly native proteins aggregate via formation of a partially unfolded intermediate. γS-Crystallin is a highly stable structural protein of the eye lens. In the mouse Opj cataract, a non-conservative F9S mutation in the N-terminal domain core of γS allows the adoption of a native fold but renders the protein susceptible to temperature- and concentration-dependent aggregation, including fibril formation. Hydrogen/deuterium exchange and denaturant unfolding studies of this mutant protein (Opj) have suggested the existence of a partially unfolded intermediate in its aggregation pathway. Here, we used NMR and fluorescence spectroscopy to obtain evidence for this intermediate. In 3.5 M urea, Opj forms a stable and partially unfolded entity that is characterized by an unstructured N-terminal domain and a largely intact C-terminal domain. Under physiologically relevant conditions, Carr-Purcell-Meiboom-Gill T(2)-relaxation dispersion experiments showed that the N-terminal domain residues were in conformational exchange with a loosely structured intermediate with a population of 1-2%, which increased with temperature. This provides direct evidence for a model in which proteins of native fold can explore an intermediate state with an increased propensity for formation of aggregates, such as fibrils. For the crystallins, this shows how inherited sequence variants or environmentally induced modifications can destabilize a well-folded protein, allowing the formation of intermediates able to act as nucleation sites for aggregation and the accumulation of light-scattering centers in the cataractous lens.

Figure 1

Overlay of 2D ¹H-¹⁵N HSQC spectra of Opj in the absence (black) and presence (red) of 3.5 M Urea recorded at 800 MHz at 25 °C. Peaks are labeled according to the assignment in the absence of denaturant. The signals of the N-terminal domain are significantly altered while those of the C-terminal domain are largely unperturbed. The three residues mentioned in the text with substantial line-broadening are colored in blue.

Figure 2

Comparison of the ¹⁵N-dispersion profiles of WT and Opj crystallins recorded at 800 (filled) and 600 (open) MHz at 32 °C. The measured effective transverse relaxation rate R₂^eff is plotted as a function of the CPMG frequency ν_cpmg. (A) Ribbon representation of γS crystallin (1ZWM) showing three groups of ¹⁵N transverse relaxation dispersion profiles with Group I color coded in red, Group II in blue and Group III in green, each represented by (B) Ile7, (C) Gln70 and (D) Lys158 with the same color scheme as (A) and black for WT protein. Each domain of γS-crystallin is constructed by two Greek-Key motifs, namely GK1 and GK2 in the N-terminal domain, and GK3 and GK4 in the C-terminal one.

Figure 3

Temperature dependence of the backbone ¹⁵N relaxation dispersion profiles collected at magnetic field strengths of 14.1 (lighter color) and 18.8 T (darker color) of (A) Cys22 from Group I, (B) Gln70 and (C) Ala84 from Group II, and (C) Lys158 from Group III of Opj, indicating both positive and negative correlation with respect to temperatures.

Figure 4

Temperature dependence of the population of the minor state of the N-terminal domain of Opj (A), the Arrhenius plot (B) and Van’t Hoff plot (C) of both the N-terminal (red) and C-terminal (green) domains. (D) Ratio of Δω_exp/Δω_cal for the backbone ¹⁵N nuclei of the N-terminal domain of Opj. Only those residues with ∣Δω_cal ∣ > 2.0 ppm have been plotted. Arrows indicate the β-stranded secondary structures.

Figure 5

ANS fluorescence upon binding to γS proteins: ANS alone (solid black), WT protein (dotted lines), and Opj mutant (solid lines) collected at 25 °C (red) and 37 °C (blue). All data were normalized according to ANS alone spectrum at 32 °C.

Figure 6

Comparison of {¹H}-¹⁵N heteronuclear NOE (top) and T_1ρ (bottom) values of Opj (red) and WT γS (black) recorded at 600 MHz at 32 °C, indicating similar ps-ns motion in both proteins. A spin lock field of 2.5 KHz with 8 durations ranging from 0.02 to 30 ms was used for T_1ρ, and a 5s-proton saturation was employed for the NOE spectrum.