Amyloid-like Properties of Bacterial Inclusion Bodies

Carrió, M. Mar; González-Montalbán, Núria; Vera, Antonio; Villaverde, Antonio; Ventura, Salvador

doi:10.1016/j.jmb.2005.02.030

Cited by 218 publications

(219 citation statements)

References 59 publications

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“…36 Rather, it results from an organized aggregation driven by interactions between hydrophobic stretches of partially folded protein molecules. 37 We hypothesize that in the case of a2N protein, its hydrophobic regions drive the formation of IBs, but it remains partially folded and, therefore, can be efficiently and quantitatively solubilized by FC-12. This finding also led us to conclude that the generally accepted ''cytosolic'' N-terminal part of a-subunit of V-ATPase is not a traditional peripheral membrane domain.…”

Section: Discussionmentioning

confidence: 97%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

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Section: Discussionmentioning

confidence: 97%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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“…The pellet was resuspended and lysed by sonication. Inclusion bodies were purified using a differential centrifugation-detergent wash procedure, 36,37 with repeated washing steps (resuspension of the pellet by sonication and centrifugation) in a buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% NaN 3 , and 0.5% triton X-100. In order to obtain a monomeric peptide solution, we employed the protocols developed by Teplow 38 and Hou et al, 39 with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

“…These are based on MAS solidstate NMR experiments, [5][6][7][8][9][10][11] and cryo-electron microscopic image reconstructions. 12 Even though there is some controversy concerning the conformational space that Ab fibrils can adopt, all published models, including the 2-fold 7,10 and 3-fold 9 symmetric Ab 1-40 fibril structure suggested by Tycko, and Bertini and co-workers, agree on the basic building block which involves a b-sheet (b1, residues 12-24), a turn, and a second b-sheet (b2, residues [28][29][30][31][32][33][34][35][36][37][38][39][40]. Biophysical studies indicate a certain degree of conformational plasticity for the N-terminal b-sheet.…”

mentioning

confidence: 99%

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

Agarwal

Linser

Dasari

et al. 2013

Phys. Chem. Chem. Phys.

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The amyloid b-peptide (Ab) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Ab peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.Alzheimer's disease (AD) is a slowly progressive neurological disorder which is associated with memory loss, cognitive impairment and finally dementia and death. The amyloid b-peptide (Ab) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Ab is a cleavage product resulting from Alzheimer Precursor Protein (APP) processing. 1 The g-secretase cut yields Ab fragments of different lengths of which Ab40 and Ab42 are most abundant. 2,3 In vitro, the Ab peptide aggregates over time into oligomers and fibrillar structures. 4 In the past, several Ab fibril structure models have been suggested. These are based on MAS solidstate NMR experiments, 5-11 and cryo-electron microscopic image reconstructions. 12 Even though there is some controversy concerning the conformational space that Ab fibrils can adopt, all published models, including the 2-fold 7,10 and 3-fold 9 symmetric Ab 1-40 fibril structure suggested by Tycko, and Bertini and co-workers, agree on the basic building block which involves a b-sheet (b1, residues 12-24), a turn, and a second b-sheet (b2, residues 28-40). Biophysical studies indicate a certain degree of conformational plasticity for the N-terminal b-sheet. 13,14 Whereas b2 is present in all the studies, b1 can be truncated or does not adopt a regular b-sheet structure. Depending on the preparation conditions, amide protons in the region of the peptide where the first b-strand is expected show differential H/D protection factors. 13 Similarly, a certain degree of conformational variability in the N-terminus is suggested using cryo-electron microscopy. 14 In all structural models, Ab peptides are arranged in a parallel fashion. An antiparallel arrangement is only observed for the Iowa mutant Ab-D23N, 15 and for truncated forms of the peptide such as Ab [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . 16 The fibril structure is stabilized in particular by hydrogen bonding interactions. Therefore, observation of exchangeable and titratable groups is of particular interest to identify hydrogen bonding patterns. We and others could show in the past that high resolution proton spectra can be obtained for...

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“…Purified recombinant IBs appear as spherical, ellipsoidal, or cylindrical particles measuring between 0.5 and 1.8 mm, characterized by a smooth and porous surface [36,37]. TsnC IBs consisted of spherical particles (Fig.…”

Section: Tsnc Ibmentioning

confidence: 99%

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

Lemke

Chura-Chambi

Rodrigues

et al. 2015

Protein Expression and Purification

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Amyloid-like Properties of Bacterial Inclusion Bodies

Cited by 218 publications

References 59 publications

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

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