Lysozyme

The cell pellets were collected from different growth phase cultures by spinning them at 13,000×g for 2–3 min at room temperature and the RNA extracted using JetGene RNA Purification Kit (Thermo Fisher Scientific). The cells were lysed with occasional vortexing in a buffer solution with 1× TE buffer, 15 mg/ml lysozyme and 20 mg/ml proteinase K (Promega). The samples were then transferred to a 2-ml Lysing Matrix A tube (MP Biomedicals) with β-mercaptoethanol containing RLT buffer (provided in the kit) for enhanced lysis. The contents in the lysing matrix tubes were then homogenised using the FastPrep™ FP 200 cell disrupter at speed 5.5 for 30 s. A double DNA-digestion treatment was done to ensure that the RNA was free of any genomic DNA (gDNA) contamination. The RNA isolated was quantified using the Nanodrop spectrophotometer with A₂₆₀/A₂₈₀ ratio of 1.8–2.1 being considered as pure. The integrity of the samples was checked by agarose gel electrophoresis for presence of two sharp distinct bands representing 23S and 16S rRNA. The integrity was further verified by analysing the samples in an Agilent 2100 Bioanalyzer where RNA Integrity Number (RIN) values greater than 8 were observed for all samples. The RIN is based on a numbering system from 1 to 10 with 1 being the most degraded and 10 being the most intact. The RNA samples were aliquoted and stored at − 80 °C.

The chaperone activity of the wild-type αB-crystallin and deletion mutant were measured using lysozyme and luciferase (Promega, Madison, WI, USA) as the aggregation substrates. The assays were carried out on a Sepctramax i3 plate reader (Molecular Devices, San Jose, CA, USA). The light scattering at 360 nm was recorded as a function of time in the presence or absence of αB-wt or αBΔ54–61. lysozyme (10 µM) aggregation assay was conducted at 37 °C in 0.25 mL PBS containing 2 mM DTT (GoldBio, St Louis, MO, USA) using 0–5 µM chaperone proteins. The luciferase (1 µM) aggregation was performed in PBS at 37 °C using 0–60 µM chaperone proteins. The relative chaperone efficiency of αB-wt and αBΔ54–61 against a substrate was compared by estimating the EC50 (effective chaperone protein concentration required to suppress the substrate protein aggregation by 50%) values. The EC50 values were calculated from the non-linear regression analysis obtained by plotting the % of substrate protein aggregation at the end of the assay for a known chaperone protein concentration. Sigmaplot V12.5 (Systat Software Inc., Palo Alto, CA, USA) dynamic curve fitting with four-parameter logistic curve function was used for non-linear curve fitting analysis.

For western blots to compare expression levels of Tha-1 and Tha-1^H270A, 1 ml of the respective overnight cultures (expressing either Tha-1 variant with an N-terminal 3xFLAG tag) was spun down, resuspended in PBS with 1 μg ml⁻¹ lysostaphin, 2 μg ml⁻¹ lysozyme and 2 μl RQ1 DNase (Promega) and incubated at 37 °C for 20 min. Samples were then run on a 15% SDS–PAGE gel with a PageRuler Plus prestained protein ladder (10–250 kDa) (ThermoFisher), transferred to a nylon membrane, blocked with 0.5% milk and human immunoglobulin G and labelled with antibodies. For Tha-1 and Tha-1^H270A, direct detection was done with a primary antibody fused to horseradish peroxidase (HRP) (at 1/1,000 dilution) against 3xFLAG. For GlmM (phosphoglucosamine mutase), a primary antibody was used first (at 1/2,500 dilution), followed by a secondary, HRP-fused anti-rabbit antibody (at 1/10,000 dilution). C-terminally His-tagged S. aureus GlmM protein was expressed and purified from Escherichia coli as previously described^{46 (link)} and used for the production of rabbit polyclonal antibodies at Covalabs under project number 1846011. The HRP substrate used was Clarity Western ECL Substrate (Bio-Rad) with a 5 min incubation time before imaging.