scholarly journals TTYH family members form tetrameric complexes within the cell membrane

2021 ◽  
Author(s):  
Emelia Melvin ◽  
Elia Shlush ◽  
Moshe Giladi ◽  
Yoni Haitin
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Size Exclusion ◽  
Cross Linking ◽  
Native Page ◽  
Intact Mouse ◽  
Detergent Solubilization ◽  
Blue Native Page ◽  
Exclusion Chromatography ◽  
Functional Analyses

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrated that mTTYH1 and mTTYH3 form tetramers at the plasma membrane. Blue-native PAGE and fluorescence-detection size-exclusion chromatography analyses revealed that detergent solubilization results in the dissolution of tetramers into dimers, suggesting a dimer-of-dimers assembly mode. As cross-linking analysis of the soluble extracellular domains also showed tetrameric stoichiometry, we explored the effect of membrane solubilization and disulfide bridges integrity and established their contribution to tetramer stability. Future studies of the native tetrameric TTYH characterized here may illuminate their long-sought cellular function.

scholarly journals The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

2002 ◽  
Vol 13 (11) ◽  
pp. 3811-3821 ◽  
Author(s):  
Pauli J. Ojala ◽  
Ville O. Paavilainen ◽  
Maria K. Vartiainen ◽  
Roman Tuma ◽  
Alan G. Weeds ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Activity ◽  
Size Exclusion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Wild Type ◽  
Native Page ◽  
Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

scholarly journals VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

2018 ◽  
Vol 115 (23) ◽  
pp. 5950-5955 ◽  
Author(s):  
Bastien Casu ◽  
Charline Mary ◽  
Aleksandr Sverzhinsky ◽  
Aurélien Fouillen ◽  
Antonio Nanci ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Secretion System ◽  
High Molecular Mass ◽  
Full Length ◽  
Size Exclusion ◽  
Cross Linking ◽  
X Ray ◽  
Exclusion Chromatography ◽  
Plasmid Pkm101 ◽  
Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

scholarly journals Single molecule fingerprinting reveals different amplification properties of α-synuclein oligomers and preformed fibrils in seeding assay.

2021 ◽  
Author(s):  
Derrick Lau ◽  
Chloe Magnan ◽  
Kathryn Hill ◽  
Antony Cooper ◽  
Yann Gambin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Amyloid Fibrils ◽  
Size Exclusion ◽  
Single Molecule Detection ◽  
Single Molecule Spectroscopy ◽  
Parkinsons Disease ◽  
Exclusion Chromatography ◽  
Particle Level ◽  
Amplification Assay ◽  
Multiple Species

The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinsons disease. However, multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify which species can be detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent but our results reveal that their seeding behaviour is very different compared to preformed fibrils in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in seeding assays.

scholarly journals Size Modulation of Enzymatically Cross-Linked Sodium Caseinate Nanoparticles via Ionic Strength Variation Affects the Properties of Acid-Induced Gels

Dairy ◽  
2021 ◽  
Vol 2 (1) ◽  
pp. 148-164
Author(s):  
Norbert Raak ◽  
Lars Leonhardt ◽  
Harald Rohm ◽  
Doris Jaros
Keyword(s):  
Molecular Structure ◽  
Ionic Strength ◽  
Structure Function ◽  
Size Exclusion Chromatography ◽  
Functional Properties ◽  
Sodium Caseinate ◽  
Size Exclusion ◽  
Cross Linking ◽  
Food Proteins ◽  
Exclusion Chromatography

Enzymatic cross-linking by microbial transglutaminase is a prominent approach to modify the structure and techno-functional properties of food proteins such as casein. However, some of the factors that influence structure-function-interrelations are still unknown. In this study, the size of cross-linked sodium caseinate nanoparticles was modulated by varying the ionic milieu during incubation with the enzyme. As was revealed by size exclusion chromatography, cross-linking at higher ionic strength resulted in larger casein particles. These formed acid-induced gels with higher stiffness and lower susceptibility to forced syneresis compared to those where the same number of ions was added after the cross-linking process. The results show that variations of the ionic milieu during enzymatic cross-linking of casein can be helpful to obtain specific modifications of its molecular structure and certain techno-functional properties. Such knowledge is crucial for the design of protein ingredients with targeted structure and techno-functionality.

scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

scholarly journals Manipulation of a cation-Π sandwich reveals conformational flexibility in phenylalanine hydroxylase

2020 ◽  
Author(s):  
Emilia C. Arturo ◽  
George Merkel ◽  
Michael R. Hansen ◽  
Sophia Lisowski ◽  
Deeanne Almeida ◽  
...  
Keyword(s):  
Phenylalanine Hydroxylase ◽  
Limited Proteolysis ◽  
Maximal Activity ◽  
Conformational Space ◽  
Size Exclusion ◽  
Protein Fluorescence ◽  
Allosteric Enzyme ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Intrinsic Protein Fluorescence

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding to the A-PAH conformation. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich between Phe80, Arg123, and Arg420, which cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared; their conformational equilibrium was evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change involving Phe80. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a C-terminal 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of the RS-PAH conformation. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH conformational interchange. Hypersensitivity is corrected by addition of Phe such that all Phe80 variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing the propensity to sample intermediates in the RS-PAH and A-PAH interchange, which are presumed on-pathway because they can readily achieve the A-PAH conformation by addition of Phe.

Intermolecular cross-linking of monomers in Helicobacter pylori Na+/H+ antiporter NhaA at the dimer interface inhibits antiporter activity

2010 ◽  
Vol 426 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Akira Karasawa ◽  
Keiji Mitsui ◽  
Masafumi Matsushita ◽  
Hiroshi Kanazawa
Keyword(s):  
Helicobacter Pylori ◽  
Conformational Changes ◽  
Cross Linking ◽  
Flanking Sequence ◽  
Native Page ◽  
Dimer Interface ◽  
Reducing Conditions ◽  
Cysteine Scanning Mutagenesis ◽  
Functional Analyses ◽  
Β Sheet

We have previously shown that HPNhaA (Helicobacter pylori Na+/H+ antiporter) forms an oligomer in a native membrane of Escherichia coli, and conformational changes of oligomer occur between monomers of the oligomer during ion transport. In the present study, we use Blue-native PAGE to show that HPNhaA forms a dimer. Cysteine-scanning mutagenesis of residues 55–61 in a putative β-sheet region of loop1 and subsequent functional analyses revealed that the Q58C mutation resulted in an intermolecular disulfide bond. G56C, I59C and G60C were found to be cross-linked by bifunctional cross-linkers. Furthermore, the Q58E mutant did not form a dimer, possibly due to electrostatic repulsion between monomers. These results imply that Gln-58 and the flanking sequence in the putative β-sheet of the monomer are located close to the identical residues in the dimer. The Q58C mutant of NhaA was almost inactive under non-reducing conditions, and activity was restored under reducing conditions. This result showed that cross-linking at the dimer interface reduces transporter activity by interfering with the flexible association between the monomers. A mutant HPNhaA protein with three amino acid substitutions at residues 57–59 did not form a dimer, and yet was active, indicating that the monomer is functional.

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