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.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

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 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 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 An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization

2019 ◽  
Vol 75 (12) ◽  
pp. 725-730 ◽  
Author(s):  
Nicole Mietrach ◽  
Andreas Schlosser ◽  
Sebastian Geibel
Keyword(s):  
Limited Proteolysis ◽  
Extracellular Domain ◽  
Secretion System ◽  
Size Exclusion ◽  
Ammonium Citrate ◽  
Diffusion Method ◽  
Space Groups ◽  
Cell Parameters ◽  
Stable Domain ◽  
Exclusion Chromatography

The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 Å resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 Å, α = β = γ = 90°.

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.

Inhibin, activin and follistatin bind preferentially to the transformed species of alpha 2-macroglobulin

1997 ◽  
Vol 155 (1) ◽  
pp. 65-71 ◽  
Author(s):  
DJ Phillips ◽  
MT Hearn ◽  
DM de Kretser ◽  
Keyword(s):  
Native Species ◽  
Binding Protein ◽  
Size Exclusion ◽  
Affinity Binding ◽  
Native Form ◽  
Native Page ◽  
Reducing Conditions ◽  
Major Species ◽  
Exclusion Chromatography ◽  
Lower Capacity

alpha 2-Macroglobulin (alpha 2-M), a major serum glycoprotein, has been implicated as a low-affinity binding protein for inhibin and activin. In serum, alpha 2-M exists as two major species, a native form that is abundant and stable, and a transformed ('fast') species that is rapidly cleared from the circulation via alpha 2-M receptors. In this study inhibin, activin and their major binding protein follistatin were investigated for their ability to bind to the native or transformed species of purified human alpha 2-M. Using native PAGE and size exclusion chromatography, radiolabelled inhibin, activin and follistatin bound to the transformed alpha 2-M. Inhibin and follistatin did not bind significantly to native alpha 2-M, whereas activin was able to bind to the native species but with a lower capacity compared with that to transformed alpha 2-M. Under reducing conditions, binding of these hormones to alpha 2-M was abolished. These findings implicate alpha 2-M as a mechanism whereby inhibin, activin and follistatin may be removed from the circulation through alpha 2-M receptors, but also whereby activin can be maintained in the circulation through its ability to bind to native alpha 2-M.

scholarly journals Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?

2007 ◽  
Vol 405 (1) ◽  
pp. 157-164 ◽  
Author(s):  
Thorsten Hornemann ◽  
Yu Wei ◽  
Arnold von Eckardstein
Keyword(s):  
Molecular Mass ◽  
De Novo ◽  
Size Exclusion ◽  
Published Data ◽  
Serine Palmitoyltransferase ◽  
Native Page ◽  
Tissue Specific ◽  
Dynamic Composition ◽  
293 Cells ◽  
Exclusion Chromatography

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1–3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.

scholarly journals X-ray diffraction analysis andin vitrocharacterization of the UAM2 protein fromOryza sativa

2017 ◽  
Vol 73 (4) ◽  
pp. 241-245 ◽  
Author(s):  
Ditte Hededam Welner ◽  
Alex Yi-Lin Tsai ◽  
Andy M. DeGiovanni ◽  
Henrik Vibe Scheller ◽  
Paul D. Adams
Keyword(s):  
Metal Ion ◽  
Limited Proteolysis ◽  
Disulfide Bridge ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
Oligomeric State ◽  
X Ray ◽  
Functional Studies ◽  
Exclusion Chromatography

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein fromOryza sativa(OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.

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