Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

Abstract

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation; however, fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.

Keywords: alphaB-crystallin; chloride intracellular channel 1; mass photometry; molecular chaperone; oligomers; protein aggregation; protein complexes; total internal reflection fluorescence microscopy.

Figure 1

αBc_WTforms high-molecular-mass complexes with CLIC1_cysL,inhibiting its amorphous aggregation.A, CLIC1_cysL (50 μM) was incubated at 37 °C for 20 h in the presence of varying molar ratios of αBc_WT (1:125–1:0.5, αBc_WT–CLIC1) or ovalbumin (Ova). Ovalbumin was used as a non-chaperone control protein at a molar ratio of 1:0.5 (CLIC1–Ova). The aggregation of CLIC1_cysL was monitored by measuring the change in light scatter at 340 nm over time. B, the percentage protection afforded by varying molar ratios of αBc_WT against CLIC1_cysL aggregation, reported as the mean ± standard deviation of three independent experiments (n = 3). C, size-exclusion chromatograms of non-incubated CLIC1_cysL (50 μM) (green), and the soluble fraction of samples following incubation; CLIC1_cysL in the absence of αBc_WT (red); αBc_WT alone (100 μM, dark blue); CLIC1_cysL in the of presence αBc_WT (lightblue, molar ratio 1:0.5, αBc_WT:CLIC1). D, SDS-PAGE of the eluted fractions collected from the size-exclusion column. The elution volume of the fractions is shown at the top of the figure.

Figure 2

αBc binds and inhibits the amorphous aggregation of CLIC1_C24by forming stable client–chaperone complexes.A, a representative aggregation assay performed to assess the ability of αBc_WT to inhibit the heat-induced aggregation of CLIC1_C24. Recombinant CLIC1_C24 was incubated at 37 °C for 20 h in the presence or absence of varying molar ratios of αBc_WT (1:0.5–1:64, αBc_WT–CLIC1_C24) or the control protein SOD1. The aggregation of CLIC1_C24 was monitored by measuring the change in light scatter at 340 nm over time. B, the percent inhibition afforded by varying molar ratios of αBc_WT against CLIC1_C24 aggregation, reported as mean ± standard deviation of three independent aggregation assays (n = 3). C, schematic of methodology used to form and surface immobilize complexes formed between AF555-CLIC1_C24 and AF647-αBc_C176 for smFRET experiments. D, representative TIRF microscopy images of AF555-CLIC1_C24 and AF647-αBc_C176 complexes. Scale bar = 5 μm. E, FRET efficiency (E) histogram derived from TIRF microscopy data of the initial intensities of CLIC1_C24–αBc_C176 complexes prior to photobleaching (n = 421 molecules). αBc, alphaB-crystallin; SOD1, superoxide dismutase 1; TIRF, total internal reflection fluorescence.

Figure 3

The binding of CLIC1 to functionalized coverslips for analysis by a single-molecule fluorescence-based approach.A–C, AF647-labeled CLIC1_C24 (1 μM) was incubated at 37 °C for 2 h before being diluted 1:1000 into imaging buffer and loaded into flow cells in the presence and absence of a surface-bound anti-6X His-tag antibody. Following a 10-min incubation, flow cells were washed and imaged using TIRF microscopy. A, representative images of surface-bound AF647-CLIC_C24 in the absence (left) or presence (right) of surface-immobilized antibodies. Scale bar = 5 μm. B, the number of CLIC1_C24 foci per field of view (FOV) on coverslips in the presence or absence of the anti-6X His-tag antibody, reported as mean ± standard deviation (n = 12). Comparisons of the treatment groups were performed via a student’s t test. C, violin plots showing the distribution of the fluorescence intensity of AF647-CLIC1_C24 foci in the presence or absence of the antibody. The plots show the kernel probability density (black outline), median (red), and interquartile range (blue). Comparisons of distributions were performed using the Kruskal–Wallis test for multiple comparisons with Dunn’s procedure. D–F, AF647-CLIC1_C24 was incubated in the presence of heated (previously for 2 h at 37 °C) or nonheated AF555-CLIC1_C24 (1 μM) for 5 min on ice. Samples were diluted 1:1000 and were loaded into flow cells before being washed and imaged using TIRF microscopy. Representative images of surface-bound (D) nonheated AF555-CLIC1_C24 (green) and AF647-CLIC1_C24 (magenta) or (E) heated AF555-CLIC1_C24 (green) and nonheated AF647-CLIC1_C24 (magenta). F, the relative abundance of each fluorescently labeled CLIC1_C24 species per FOV reported as mean ± standard deviation (n = 15). TIRF, total internal reflection fluorescence.

Figure 4

Characterization of CLIC1_C24–αBc_C176complexes using a single-molecule fluorescence-based approach. AF488-αBc_C176 was incubated with AF647-CLIC1_C24 (2:1 molar ratio) at 37 °C for 10 h to form complexes. Aliquots were taken at multiple timepoints throughout the incubation for TIRF microscopy imaging. A, representative TIRF microscopy images of complexes at 10 h. Scale bar = 5 μm. Schematic indicating free CLIC1_C24 and αBc_C176 bound to the coverslip surface. B, schematic showing the immobilization of αBc_C176–CLIC1_C24 complexes to the surface of a glass coverslip. The percentage of CLIC1_C24 colocalized with αBc_C176 over time reported as the mean ± standard deviation of three independent experiments. Data were fit using a one-phase association model. C, example time trace of the fluorescent intensity of AF647-CLIC1_C24 in complex with AF488-αBc_C176. The shaded area (gray) represents the first 20 values that were averaged to determine the initial intensity (I₀). D, photobleaching traces from AF647-CLIC1_C24 molecules with distinct photobleaching steps were manually identified and fit to a change point analysis to calculate the fluorescent intensity of each single-photobleaching event (I_s). The I_s values were fit to a Gaussian distribution to determine the mean intensity of a single photobleaching event (I_s-mean). E, example histogram of CLIC1_C24 showing the distribution of I₀ and fluorescently labeled proteins per point (FPP) at 10 h. FPP were calculated using the equation FPP = I₀/I_s-mean for all the CLIC1_C24 in complex with αBc_C176. TIRF, total internal reflection fluorescence.

Figure 5

αBc_C176-CLIC1_C24complexes increase in polydispersity and size over time. AF488-αBc_C176 was incubated with AF647–CLIC1_C24 (2:1 molar ratio) at 37 °C for 10 h, with aliquots taken at multiple timepoints throughout the incubation. Following incubation, aliquots were immediately diluted and incubated in flow cells for 10 min before being washed and imaged using TIRF microscopy. Violin plots showing the size distribution over 10 h at 37° of (A) free CLIC1_C24 that is not in complex with αBc_C176, (B) CLIC1_C24 bound to αBc_C176 or free CLIC1_C24 after 0.25 h of incubation, (C) CLIC1_C24 bound to αBc_C176, (D) CLIC1_C24 bound to αBc_C176 or free CLIC1_C24 after 10 h of incubation, (E) αBc_C176 bound to CLIC1_C24, and (F) αBc_C176 bound to CLIC1_C24 or nonspecifically adsorbed to the surface (Free) after 10 h. The violin plots show the kernel probability density (black outline), median (red), and interquartile range (blue). Results include measurements from three independent experiments (n = 3), and comparisons of distributions were performed using the Kruskal–Wallis test for multiple comparisons with Dunn’s procedure (p values indicated). G, heatmaps showing the relative abundance of αBc_C176–CLIC1_C24 complexes and their stoichiometries over 8 h of incubation. TIRF, total internal reflection fluorescence.

Figure 6

αBc_C176–CLIC1_C24complexes change in size and stoichiometry with increasing αBc_C176concentration. AF647-CLIC1_C24 was incubated in the presence of varying molar ratios of AF488-αBc_C176 at 37 °C for 8 h. Following incubation, samples were immediately diluted and incubated in flow cells for 10 min before being washed and imaged using TIRF microscopy. The size distributions of CLIC1_C24 (A) in complex with αBc_C176 (B) at increasing molar ratios of αBc_C176–CLIC1_C24. The violin plots show the kernel probability density (black outline), median (red), and interquartile range (blue). Result are representative of two independent experiments (n = 2), and comparisons of distributions were performed using the Kruskal–Wallis test for multiple comparisons with Dunn’s procedure (p values indicated). C, heatmaps showing the relative abundance of αBc_c176–CLIC1_C24 complexes with increasing molar ratios of αBc_C176–CLIC1_C24. TIRF, total internal reflection fluorescence.

Figure 7

Schematic of two-step mechanism of sHsp–client complex formation.A, smaller free sHsps initially recognize and stably bind free misfolded client proteins (1) allowing for subsequent binding of additional free sHsps subunits to form larger sHsp–client complexes (2). B, theoretical binding events of sHsp subunits over time showing that initial binding of free sHsps to free clients increases over time (1) until all the misfolded client is bound and additional free sHsp subunits associate with these complexes (2) in order to form larger sHsp–client complexes. sHsp, small heat shock protein.