FIGURE 2.

A, 500 MHz ¹H NMR spectra of an ordinary sample of R3Aβ2m (∼0.9 mm, 310 K, pH 6.6) in the absence (lower trace) and presence (upper trace) of an equimolar amount of αB-crystallin subunit. The only apparent consequence of the sHsp addition is a general broadening, with conservation of the spectral pattern of R3Aβ2m. The insets show a portion of the H^N-H^α connectivity (fingerprint) region of the corresponding two-dimensional TOCSY spectra. The boxed cross-peaks arise from the unstructured and flexible C-terminal extension of αB-crystallin. Only this region of 12 amino acids is present in the spectrum by virtue of its fast local tumbling as opposed to the extremely slow overall motion of αB-crystallin oligomer (∼650 kDa) that causes the extreme broadening of other resonances (34–36, 60). Assignments are indicated for R3Aβ2m NH to α-CH cross-peaks (12, 19). Arrows mark visibly attenuated R3Aβ2m connectivities for Q69 and T86. B, time evolution of the resolved upfield-shifted methyl resonance (L23 δ₁CH₃), which is diagnostic for the correctly folded species, of an ordinary sample of R3Aβ2m obtained from 500 MHz ¹H one-dimensional NMR spectra, in the absence (circles) and in the presence of αB-crystallin at equimolar (squares) or halved concentration (triangles) with respect to R3Aβ2m. The average rate of loss of signal intensity was (5.6 ± 0.5) × 10⁻³ h⁻¹ in the absence of αB-crystallin and (1.5 ± 0.5) × 10⁻³ or (1.8 ± 0.5) × 10⁻³ h⁻¹ for 1:2 or 1:1 αB-crystallin/R3Aβ2m mixtures, respectively. The errors on the rates were estimated by considering the deviations obtained when assessments were made using absolute resonance amplitudes rather than self-normalized values (53) to account for the uncertainties introduced by the scaling procedure. Generally speaking, such uncertainties arise when resonances with smaller linewidths overlap pre-existing signals, as well as from the general linewidth increase upon addition of αB-crystallin. C, two-dimensional TOCSY fingerprint details obtained from an 82-h-old solution of R3Aβ2m (0.8 mm, 310 K, pH 6.6). The boxed cross-peaks represent the NH-αCH connectivities whose time evolution is displayed in D, respectively, from Phe-30, in the folded protein, and from an unknown residue, in the unfolded soluble species. D, time dependence of the two-dimensional TOCSY cross-peak amplitudes of C (Phe-30 = squares, unassigned = diamonds). In the absence of αB-crystallin, the evolution of the resolved fingerprint cross-peaks showed typically a similar trend as that obtained by considering the evolution of the resolved methyl resonances in one-dimensional ¹H spectra (B). Indeed, the slope of the decreasing Phe-30 cross-peak amplitude is −5.9 × 10⁻³ h ⁻¹, i.e. in absolute value the same, within the experimental error, as the slope of the unknown cross-peak intensity.