Figure 1. Suppression of human γD-crystallin aggregation by myo-inositol.

Oxidative aggregation of the cataract-mimicking W42Q variant of HγD was initiated as previously described (Serebryany et al., 2018). (A) Normalized turbidity traces for the oxidative aggregation of 40 μM HγD W42Q with varying concentrations of myo-inositol. (B) Sugars and sugar alcohols, each at 100 mM, suppressed HγD W42Q aggregation to varying degrees. Isopropyl-β-D-thiogalactoside (IPTG) moderately enhanced turbidity development. Myo-inositol (structure shown in inset) consistently and strongly suppressed turbidity development, second only to the disaccharides, trehalose and sucrose. A total of 8 independent replicates, at pH 7.4 (HEPES), pH 6.7 (PIPES), or pH 6.0 (MES) and 150 mM NaCl all produced similar results and were averaged together. Notably, the strong linear correlation between reduction in solution turbidity and increase in the proportion of protein remaining soluble indicated these compounds (except perhaps IPTG) did not significantly change aggregate geometry. Raw data are available as Figure 1—source data 1. (C) Dose response for aggregation suppression by myo-inositol compared to glycerol. Notably, myo-inositol had a significant effect in the physiological concentration range. Data from two independent replicates (circles, squares) are shown; black circles correspond to the data in panel A. All aggregation rates were normalized to the rate without myo-inositol. (D) Percent change in aggregation lag time for the same experiments as in Panel C. (E) Suppression of oxidative aggregation by myo-inositol generalizes to other HγD constructs: Green triangles, 20 μM W42Q whose aggregation is catalyzed by 180 μM WT HγD at 37 °C; Beige circles, 40 μM W130Q at 42 °C; Purple diamonds, 50 μM wild-type isolated N-terminal domain of HγD at 44 °C. All experiments were carried out in 10 mM PIPES pH 6.7, 150 mM NaCl, 1 mM EDTA, with 0.5 mM GSSG as the oxidant. Raw data and fitting for panels C,D,E are available as Figure 1—source data 2.

Figure 1—figure supplement 1. 100 mM inositol does not significantly affect bulk solution properties.

Solution rheometry was used to measure viscosity of the aqueous buffer (10 mM PIPES, pH 6.7) with and without 100 mM myo-inositol or select other compounds. No meaningful difference was observed between 100 mM myo-inositol and plain buffer. Changes produced by the other compounds were minor. As a positive control, 20% w/v glucose is shown; this concentration (~1.1 M) is more typical of the range in which stabilizing effects of protective osmolytes are observed.

Figure 1—figure supplement 2. Comparison of myo- and scyllo-inositol.

Both compounds were tested in parallel for their ability to suppress aggregation of the W42Q HγD variant, exactly as in Figure 1A. However, scyllo-inositol’s solubility is an order of magnitude lower than that of the more common myo- isomer, which strictly constrained the testable concentration range. In this range, no significant difference was observed between myo- and scyllo-inositol. The axes are scaled to match Figure 1C and D; the inset shows a zoom-in of the data series. All aggregation rates are expressed as % of the rate without inositol.

Figure 1—figure supplement 3. Raman spectroscopy shows no signature of increased inositol self-association between 100 and 500 mM.

(A) Baseline-subtracted low-wavenumber Raman spectra showed peaks at 892 and 1071 cm^–1 for inositol in water shifted by –45 and –39 cm^–1, respectively, in D₂O (black arrows), which suggests they are reporter peaks for inositol-water hydrogen bonding. This range includes C—O and C—C—O vibrational modes of alcohols (see Larkin, P. J. Infrared and raman spectroscopy: principles and spectral interpretation. Elsevier, 2011.) The peaks at 423 and 504 cm^–1 gained intensity in D₂O but showed a much smaller shift (–8 cm^–1); we tentatively assign them to C—C vibrational modes within the inositol molecule. (B) Low-wavenumber Raman spectral readouts (300–1100 cm^–1) of inositol in water did not show any new feature emerging as the inositol concentration increased from 100 to 500 mM, only increasing intensity of both solvent-sensitive and solvent-insensitive peaks. This indicates no significant change in physico-chemical properties that would have been expected from direct inositol-inositol hydrogen bonding (or other self-association) across the concentrations tested.

Figure 1—figure supplement 4. Aggregation of W130Q and isolated WT N-terminal domain is completely redox-dependent.

The proteins were aggregated in 10 mM PIPES pH6.7, 150 mM NaCl buffer with 0.5 mM GSSG and 1 mM EDTA, at the temperature and concentrations shown, with or without 100 mM myo-inositol. In the control reactions, 0.5 mM GSSG was replaced with 1 mM DTT, resulting in no detectable aggregation (blue traces).