The oligomer plasticity of the small heat-shock protein Lo18 from Oenococcus oeni influences its role in both membrane stabilization and protein protection

2012 ◽  
Vol 444 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Magali Maitre ◽  
Stéphanie Weidmann ◽  
Aurélie Rieu ◽  
Daphna Fenel ◽  
Guy Schoehn ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Dynamic Equilibrium ◽  
Building Blocks ◽  
Oenococcus Oeni ◽  
Size Exclusion ◽  
Acidic Ph

The ability of the small Hsp (heat-shock protein) Lo18 from Oenococcus oeni to modulate the membrane fluidity of liposomes or to reduce the thermal aggregation of proteins was studied as a function of the pH in the range 5–9. We have determined by size-exclusion chromatography and analytical ultracentrifugation that Lo18 assembles essentially as a 16-mer at acidic pH. Its quaternary structure evolves to a mixture of lower molecular mass oligomers probably in dynamic equilibrium when the pH increases. The best Lo18 activities are observed at pH 7 when the particle distribution contains a major proportion of dodecamers. At basic pH, particles corresponding to a dimer prevail and are thought to be the building blocks leading to oligomerization of Lo18. At acidic pH, the dimers are organized in a double-ring of stacked octamers to form the 16-mer as shown by the low-resolution structure determined by electron microscopy. Experiments performed with a modified protein (A123S) shown to preferentially form dimers confirm these results. The α-crystallin domain of Methanococcus jannaschii Hsp16.5, taken as a model of the Lo18 counterpart, fits with the electron microscopy envelope of Lo18.

Coiled-coil deformations in crystal structures: themeasles virusphosphoprotein multimerization domain as an illustrative example

2014 ◽  
Vol 70 (6) ◽  
pp. 1589-1603 ◽  
Author(s):  
David Blocquel ◽  
Johnny Habchi ◽  
Eric Durand ◽  
Marion Sevajol ◽  
François Ferron ◽  
...  
Keyword(s):  
Measles Virus ◽  
Quaternary Structure ◽  
Dynamic Equilibrium ◽  
Crystal Packing ◽  
Coiled Coil ◽  
Terminal Region ◽  
Size Exclusion ◽  
Structural Deformation ◽  
Structural Comparison ◽  
Exclusion Chromatography

The structures of two constructs of themeasles virus(MeV) phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communieet al.(2013),J. Virol.87, 7166–7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.

scholarly journals Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

2014 ◽  
Vol 80 (10) ◽  
pp. 2973-2980 ◽  
Author(s):  
Magali Maitre ◽  
Stéphanie Weidmann ◽  
Florence Dubois-Brissonnet ◽  
Vanessa David ◽  
Jacques Covès ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Phospholipid Composition ◽  
Small Heat Shock Protein ◽  
Oenococcus Oeni ◽  
Lipid Binding ◽  
Phospholipid Content ◽  
Ethanol Stress ◽  
Content Type

ABSTRACTMalolactic fermentation in wine is often carried out byOenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response inO. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared fromO. oenigrown under optimal growth conditions (here, control liposomes), one was prepared fromO. oenigrown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids fromBacillus subtilisorLactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes fromO. oenisuggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation ofO. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.

scholarly journals Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity

2004 ◽  
Vol 381 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Tirumala Kumar CHOWDARY ◽  
Bakthisaran RAMAN ◽  
Tangirala RAMAKRISHNA ◽  
Chintalagiri Mohan RAO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Quaternary Structure ◽  
Citrate Synthase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Nucleotide Sequence Analysis ◽  
Heat Shock Factor 1 ◽  
Induced Aggregation

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic α-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

scholarly journals Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains

2015 ◽  
Vol 71 (4) ◽  
pp. 986-995 ◽  
Author(s):  
C. M. D. Swarbrick ◽  
M. A. Perugini ◽  
N. Cowieson ◽  
J. K. Forwood
Keyword(s):  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Functional Characterization ◽  
Fatty Acyl ◽  
Size Exclusion ◽  
X Ray Scattering ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Enzyme Families

Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on theEscherichia colistructure.Yersinia pestis, a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome; here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of theY. pestisTesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.

scholarly journals Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase fromBacteroides fragilis

2014 ◽  
Vol 70 (9) ◽  
pp. 1206-1210 ◽  
Author(s):  
Chongyun Cheng ◽  
Jianhua Gu ◽  
Jing Su ◽  
Wei Ding ◽  
Jie Yin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Analytical Ultracentrifugation ◽  
Structural Information ◽  
Three Dimensional ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Bifunctional Enzyme ◽  
X Rays ◽  
Cell Parameters ◽  
Cryo Electron Microscopy

Fucokinase/L-fucose-1-P-guanylyltransferase (FKP) is a bifunctional enzyme which converts L-fucose to Fuc-1-P and thence to GDP-L-fucose through a salvage pathway. The molecular weights of full-length FKP (F-FKP) and C-terminally truncated FKP (C-FKP, residues 300–949) are 105.7 and 71.7 kDa, respectively. In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size-exclusion chromatography experiments and analytical ultracentrifugation results showed that both F-FKP and C-FKP are trimers. Native F-FKP protein was crystallized by the vapour-diffusion method and the crystals belonged to space groupP212121and diffracted synchrotron X-rays to 3.7 Å resolution. The crystal unit-cell parameters area= 91.36,b= 172.03,c= 358.86 Å, α = β = γ = 90.00°. The three-dimensional features of the F-FKP molecule were observed by cryo-EM (cryo-electron microscopy). The preliminary cryo-EM experiments showed the F-FKP molecules as two parallel disc-shaped objects stacking together. Combining all results together, it is assumed that there are six FKP molecules in one asymmetric unit, which corresponds to a calculated Matthews coefficient of 2.19 Å3 Da−1with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.

scholarly journals Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp)

2021 ◽  
Vol 22 (15) ◽  
pp. 7777
Author(s):  
Lydia K. Muranova ◽  
Vladislav M. Shatov ◽  
Andrey V. Slushchev ◽  
Nikolai B. Gusev
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Quaternary Structure ◽  
Small Heat Shock Protein ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Wild Type ◽  
Simple Method ◽  
Exclusion Chromatography ◽  
Shock Proteins

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17–29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the β7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

scholarly journals One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

2004 ◽  
Vol 279 (50) ◽  
pp. 52338-52345 ◽  
Author(s):  
Chia-Hui Chien ◽  
Li-Hao Huang ◽  
Chi-Yuan Chou ◽  
Yuan-Shou Chen ◽  
Yu-San Han ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Dynamic Equilibrium ◽  
Intact Cell ◽  
Size Exclusion ◽  
Wild Type ◽  
Site Mutation ◽  
Dimer Formation ◽  
Dpp Iv ◽  
Terminal Loop

DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell andin vitroafter purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease inkcat. Replacement of His750with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.

Sign in / Sign up

Export Citation Format

Share Document