In Vitro Structural and Functional Characterization of the Small Heat Shock Proteins (sHSP) of the Cyanophage S-ShM2 and Its Host, Synechococcus sp. WH7803

Abstract

We previously reported the in silico characterization of Synechococcus sp. phage 18 kDa small heat shock protein (HspSP-ShM2). This small heat shock protein (sHSP) contains a highly conserved core alpha crystalline domain of 92 amino acids and relatively short N- and C-terminal arms, the later containing the classical C-terminal anchoring module motif (L-X-I/L/V). Here we establish the oligomeric profile of HspSP-ShM2 and its structural dynamics under in vitro experimental conditions using size exclusion chromatography (SEC/FPLC), gradient native gels electrophoresis and dynamic light scattering (DLS). Under native conditions, HspSP-ShM2 displays the ability to form large oligomers and shows a polydisperse profile. At higher temperatures, it shows extensive structural dynamics and undergoes conformational changes through an increased of subunit rearrangement and formation of sub-oligomeric species. We also demonstrate its capacity to prevent the aggregation of citrate synthase, malate dehydrogenase and luciferase under heat shock conditions through the formation of stable and soluble hetero-oligomeric complexes (sHSP:substrate). In contrast, the host cyanobacteria Synechococcus sp. WH7803 15 kDa sHSP (HspS-WH7803) aggregates when in the same conditions as HspSP-ShM2. However, its solubility can be maintained in the presence of non-ionic detergent Triton™X-100 and forms an oligomeric structure estimated to be between dimer and tetramer but exhibits no apparent inducible structural dynamics neither chaperon-like activity in all the assays and molar ratios tested. SEC/FPLC and thermal aggregation prevention assays results indicate no formation of hetero-oligomeric complex or functional interactions between both sHSPs. Taken together these in vitro results portray the phage HspSP-ShM2 as a classical sHSP and suggest that it may be functional at the in vivo level while behaving differently than its host amphitropic sHSP.

Fig 1. Migration of purified HspSP-ShM2 (18 kDa) and HspS-WH7803 (15 kDa) on SDS-PAGE.

2 μg of purified sHSPs were loaded onto 12% SDS-PAGE to examine purity and MW. The gel was stained with Coomassie Brilliant Blue G-250.

Fig 2. SEC and native gel analysis of HspSP-ShM2 and HspS-WH7803 oligomeric species.

Each sHSP was analyzed at two different concentrations, 30 μM (solid line) and 60 μM (dotted line). A) HspSP-ShM2 first peak (*) elution volume is equivalent to ~ 600 kDa (32mer) and the second one (**) to ~ 200 kDa (dodecamer). B) HspS-WH7803 peak elution volume is equivalent to ~ 60 kDa (tetramer). C) Novex™ 4–20% Tris-Glycine native gels were used to compare the oligomeric profiles of HspSP-ShM2 and HspS-WH7803. The elution volumes of protein molecular weight standards are shown in the left panel.

Fig 3. DLS analysis of HspSP-ShM2 oligomers and HspSP-ShM2 but not HspS-WH7803 dissociates into a suboligomeric species when heated.

A) Scattered intensity recorded as a function of time for increasing temperatures. B) Proportion of HspSP-ShM2 Rh at 25°C. C) Evolution of Rh with the corresponding standard deviation (polydispersity) at different temperatures. D) To assess the effect of constant heat on structural dynamics, HspSP-ShM2 or HspS-WH7803 were loaded into a 4–20% Tris-Glycine native gel and separated either at 25°C or pre-incubated 15 min and ran at 45°C.

Fig 4. HspSP-ShM2 and HspWH7803 do not form hetero-oligomers at different concentrations of Triton™ X-100, different molar ratios nor under heat treatment.

A and B) HspSP-ShM2 (dotted line) and HspS-WH7803 (dashed line) were incubated 30 min either alone or together (solid line) at a molar ratio of 1:1 at two different concentrations of Triton X-100™, (A) 0.23 mM and (B) 0.12 mM. Note that elution peaks of mixed sHSPs conditions for both detergent concentrations are equal as the ones of sHSPs alone. Protein elution volume was monitored by absorbance at 280 nm. The elution volumes of protein molar weight standards are shown above the first panel. C, D and E) Novex™ 4–20% Tris-Glycine native gels were used to compare the oligomeric profiles of (C) HspSP-ShM2 and HspS-WH7803 alone as controls for subsequent comparison (D) HspSP-ShM2 and HspS-WH7803 mixed together at different molar ratios and (E) HspS-ShM2 and HspS-WH7803 mixed together at an equimolar ratio, heat shocked at the indicated temperature, cooled down and migrated at room temperature (~25°C). The migration distances of protein molecular weight standards are shown between the panels C and D.

Fig 5. HspSP-ShM2 inhibits heat-induced aggregation of protein clients.

A, B and C) CS (A), MDH (B) and Luc (C) were incubated alone or in presence of HspSP-ShM2 at the indicated molar ratios (sHSP:Client). D) Luc was incubated either alone (Luc) or with HspS-WH7803 at the indicated molar ratio. E) MDH was incubated with HspS-WH7803 12:1 (2) and CS was incubated alone (CS), with HspS-WH7803 10:1 (3) or with HspSP-ShM2 at two molar ratios 10:1 (4) and 5:1 (5). F) CS was incubated either alone (CS), in presence of HspSP-ShM2 5:1 (3) and 10:1 (5) or in the presence of HspSP-ShM2 and HspS-WH7803 5:5:1 (4) and 10:10:1 (6).

Fig 6. HspSP-ShM2 maintains heat stressed protein clients soluble and form stable complexes.

A and B) Luc and MDH clients were incubated either alone or in presence of HspS-ShM2 at the indicated molar ratios. Both Luc and MDH fully aggregate in absence of HspSP-ShM2 as shown by their presence in the pellet fraction while both are maintained soluble when in presence of HspSP-ShM2. C) MDH and HspSP-ShM2 were incubated for 60 min at 45°C (dotted line) or at room temperature (solid line) at a 12:1 (sHSP:MDH) molar ratio and D) Luc and HspSP-ShM2 were incubated for 8 min at 42°C (dotted line) or at room temperature (solid line) at a 12:1 (sHSP:Luc) molar ratio. Note that HspSP-ShM2 forms complexes with MDH and Luc when heated (dotted line) since the peaks (*) have a lower elution volume, therefore representing a higher molecular weight than HspSP-ShM2 alone (**). Moreover, the peak representing MDH when not heated (C) (***) completely disappears after heat treatment. The elution volumes of protein molecular weight standards are shown above the panel.