Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (Ta CT-ACD and Ps CT-ACD) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the Ta CT-ACD and Ps CT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Ta dimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHspsubstrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.
Small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as ATP-independent molecular chaperones to prevent irreversible aggregation of stress-labile proteins. sHSPs range in size from ~12 to 42 kDa, but typically assemble into 12 to >32 subunit oligomers. The monomers are defi ned by a conserved α-crystallin domain fl anked by divergent and fl exible N-terminal and C-terminal arms. In higher plants sHSPs have evolved independently of metazoan and bacterial homologs and comprise multiple families of cytosolic proteins, along with proteins targeted to the nucleus, chloroplasts, mitochondria, endoplasmic reticulum and peroxisomes. This diversity of sHSPs is unique to land plants and likely arose as a result of their frequent exposure to stress due to their sessile nature. The availability of the high resolution structure of a dodecameric cytosolic class I sHSP from wheat, Ta16.9 (PDB ID: 1GME; 2.7 Å resolution), has facilitated detailed in vitro studies of sHSP chaperone action. A working model proposes that sHSP oligomers dissociate into dimers during heat stress, revealing hydrophobic patches that interact with exposed hydrophobic regions on denaturing substrates, maintaining them in a soluble, folding-competent state. sHSP-substrate complexes are then acted on by ATP-dependent chaperones to restore substrates to their native state. However, much remains to be done to connect this model with the function of the many different sHSPs found in plants. Further genetic and biochemical studies are needed to identify sHSP substrates and to defi ne the mechanism by which sHSPs function, not only during stress, but also during specifi c developmental stages in plants.
The inducible carbon concentration mechanism (CCM) in Chlamydomonas reinhardtii has been well defined from a molecular and ultrastructural perspective. Inorganic carbon transport proteins, and strategically located carbonic anhydrases deliver CO2 within the chloroplast pyrenoid matrix where Rubisco is packaged. However, there is little understanding of the fundamental signalling and sensing processes leading to CCM induction. While external CO2 limitation has been believed to be the primary cue, the coupling between energetic supply and inorganic carbon demand through regulatory feedback from light harvesting and photorespiration signals could provide the original CCM trigger. Key questions regarding the integration of these processes are addressed in this review. We consider how the chloroplast functions as a crucible for photosynthesis, importing and integrating nuclear-encoded components from the cytoplasm, and sending retrograde signals to the nucleus to regulate CCM induction. We hypothesize that induction of the CCM is associated with retrograde signals associated with photorespiration and/or light stress. We have also examined the significance of common evolutionary pressures for origins of two co-regulated processes, namely the CCM and photorespiration, in addition to identifying genes of interest involved in transcription, protein folding, and regulatory processes which are needed to fully understand the processes leading to CCM induction.
Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and their mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show that substructural dimers of plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and non-dimeric, to restrict how their assemblies can dissociate. When disulfide bonded at the non-dimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive, but when reduced had wild-type-like chaperone activity, demonstrating that dissociation at non-dimeric interfaces is essential for activity. In addition, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different than the Ta16.9 x-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) showed greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Size and secondary structure changes with temperature revealed that folded sHsp dimers interact with substrate, and support dimer stability as a determinant of chaperone efficiency. These data yield a model in which sHsp dimers capture substrate prior to assembly into larger, heterogeneous sHSP-substrate complexes for subsequent substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.
A nonredundant data set having 5025 polypeptides with 2311 disulfides was used to study cross‐strand disulfides (CSDs) in antiparallel beta‐sheets, and intrahelical disulfides (IHDs).75 and 1 CSDs were found at non‐hydrogen‐bonded (NHB) and hydrogen‐bonded (HB) registered pairs, respectively. Disulfides at HB pairs lead to steric repulsions with main chain and require a rare, positive χ1 value for at least one of the Cys. 13 disulfides were introduced in NHB and HB pairs in 4 model proteins: Leu binding protein (LBP), Leu, Ile, Val binding protein (LIVBP), maltose binding protein (MBP), and Top7. The LIVBP mutant did not show disulfide formation. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the exposed NHB LBP mutant showed an increase of 3.1 °C in Tm. Both exposed and 2 of the 3 buried NHB Top7 mutants were stabilized. All 4 HB Top7 mutants were destabilized (ΔΔGo = −3.3 to −6.7 kcal/mol). Thus, CSDs engineered at exposed NHB pairs can be used to improve protein stability.35 examples were found of IHDs between the N‐Cap and 3rd helical positions. Cys pairs were introduced at N‐Cap‐3; 1,4; 7,10 in 2 helices of an E. coli thioredoxin (Trx) mutant. Disulfides spontaneously formed only at positions N‐Cap‐3 during purification. All oxidized and reduced mutants were destabilized relative to nSS Trx. All mutants were mildly redox active.
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