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.
Highlights
The small heat shock proteins are ubiquitous ATP-independent molecular chaperones participating in many cellular processes but mainly characterized for their role in proteostasis [1,2,3]
Cyanophage HspSP-ShM2 was shown to form two main oligomers by Size Exclusion Chromatography (SEC), the latter species seeming to be concentration dependent, whereas native gels electrophoresis and dynamic light scattering (DLS) displayed an overall shift of oligomeric structures and a more polydisperse profile
The use of different methods to assess protein structural dynamic and identification of structural entities has proven to reveal different results depending on the sensitivity and measurement methodology of the experimental technique
Summary
The small heat shock proteins (sHSPs) are ubiquitous ATP-independent molecular chaperones participating in many cellular processes but mainly characterized for their role in proteostasis [1,2,3] They are composed of highly variable N- (NTR) and C-terminal (CTR) regions flanking the conserved ~ 90 amino acids α-crystallin domain (ACD) and have an average molecular masses of 12-kDa to 42-kDa [4, 5]. They are either present in one main oligomeric form (monodispersity; [6, 7]) or in a variety of oligomeric forms with different abundance (polydispersity; [8,9,10,11]) reflecting a high dynamism in quaternary structure conformation as a result of rapid and extensive subunit exchange between oligomers Under stress conditions such as heat, sHSPs chaperone activity prevents irreversible aggregation maintaining protein clients solubility by binding hydrophobic exposed surfaces while in process of stress-induced denaturation [12]. By increasing protein/lipid ratio thereby modulating fluidity and microviscosity, amphitropic sHSPs allow conservation of membrane integrity under heat stress conditions [24,25,26,27,28]
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