Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites

2002 ◽  
Vol 361 (2) ◽  
pp. 307-315 ◽  
Author(s):  
María GASSET ◽  
Carlos ALFONSO ◽  
José L. NEIRA ◽  
Germán RIVAS ◽  
María A. PAJARES
Keyword(s):  
Free Energy ◽  
Rat Liver ◽  
Active Sites ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Methionine Adenosyltransferase ◽  
Size Exclusion ◽  
Activity Measurements ◽  
Exclusion Chromatography ◽  
Monomeric Intermediate

The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24±0.15. The variations of free energy in the absence of denaturant [ΔG(H2O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15±2.3kJ·mol−1 and 19.87±0.71kJ·mol−1·M−1 for the dissociation of the native dimer and 14.77±1.63kJ·mol−1 and 5.23±0.21kJ·mol−1·M−1 for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69kJ·mol−1 and 30.33kJ·mol−1·M−1 respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established.

scholarly journals Characterisation of mass distributions of solvent-fractionated lignins using analytical ultracentrifugation and size exclusion chromatography methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yudong Lu ◽  
Lionard Joosten ◽  
Jacqueline Donkers ◽  
Fabrizio Andriulo ◽  
Ted M. Slaghek ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Molar Mass ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Compositional Analysis ◽  
Size Exclusion ◽  
Sedimentation Equilibrium ◽  
Average Molar Mass ◽  
Exclusion Chromatography ◽  
Reliable Knowledge

AbstractLignins are valuable renewable resources for the potential production of a large array of biofuels, aromatic chemicals and biopolymers. Yet native and industrial lignins are complex, highly branched and heterogenous macromolecules, properties that have to date often undermined their use as starting materials in lignin valorisation strategies. Reliable knowledge of weight average molar mass, conformation and polydispersity of lignin starting materials can be proven to be crucial to and improve the prospects for the success of such strategies. Here we evaluated the use of commonly-used size exclusion chromatography (SEC)—calibrated with polystyrene sulphonate standards—and under-used analytical ultracentrifugation—which does not require calibration—to characterise a series of lignin fractions sequentially extracted from soda and Kraft alkaline lignins using ethyl acetate, methyl ethyl ketone (MEK), methanol and acetone:water (fractions F01–F04, respectively). Absolute values of weight average molar mass (Mw) determined using sedimentation equilibrium in the analytical ultracentrifuge of (3.0 ± 0.1) kDa and (4.2 ± 0.2) kDa for soda and Kraft lignins respectively, agreed closely with previous SEC-determined Mws and reasonably with the size exclusion chromatography measurements employed here, confirming the appropriateness of the standards (with the possible exceptions of fraction F05 for soda P1000 and F03 for Indulin). Both methods revealed the presence of low (~ 1 kDa) Mw material in F01 and F02 fractions followed by progressively higher Mw in subsequent fractions. Compositional analysis confirmed > 90% (by weight) total lignins successively extracted from both lignins using MEK, methanol and acetone:water (F02 to F04). Considerable heterogeneity of both unfractionated and fractionated lignins was revealed through determinations of both sedimentation coefficient distributions and polydispersity indices. The study also demonstrates the advantages of using analytical ultracentrifugation, both alongside SEC as well as in its own right, for determining absolute Mw, heterogeneity and conformation information for characterising industrial lignins.

scholarly journals Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Morgane Agez ◽  
Elodie Desuzinges Mandon ◽  
Thomas Iwema ◽  
Reto Gianotti ◽  
Florian Limani ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Analytical Ultracentrifugation ◽  
B Cell Lymphoma ◽  
Integral Membrane Protein ◽  
Size Exclusion ◽  
Hek293 Cells ◽  
Extraction And Purification ◽  
Exclusion Chromatography ◽  
Specificity Of Binding

Abstract CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

scholarly journals Homodimerization of calpain 3 penta-EF-hand domain

2005 ◽  
Vol 388 (2) ◽  
pp. 585-591 ◽  
Author(s):  
Ravikiran RAVULAPALLI ◽  
Beatriz GARCIA DIAZ ◽  
Robert L. CAMPBELL ◽  
Peter L. DAVIES
Keyword(s):  
Skeletal Muscle ◽  
Molecular Modelling ◽  
Analytical Ultracentrifugation ◽  
Domain Size ◽  
Small Subunit ◽  
Size Exclusion ◽  
Relative Molecular Mass ◽  
Calpain 3 ◽  
Ef Hand ◽  
Exclusion Chromatography

Calpains 1 and 2 are heterodimeric proteases in which large (relative molecular mass Mr 80000) and small (Mr 28000) subunits are linked through their respective PEF (penta-EF-hand) domains. The skeletal muscle-specific calpain 3 is believed not to form a heterodimer with the small subunit but might homodimerize through its PEF domain. Size-exclusion chromatography and analytical ultracentrifugation of the recombinant PEF domain of calpain 3 show that it forms a stable homodimer that does not dissociate on dilution. Molecular modelling suggests that there would be no barriers to the dimerization of the whole enzyme through the PEF domains. This orientation would place the catalytic centres at opposite ends of the dimer.

Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes

2010 ◽  
Vol 429 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Andrew Niewiarowski ◽  
Alison S. Bradley ◽  
Jayesh Gor ◽  
Adam R. McKay ◽  
Stephen J. Perkins ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Adenine Nucleotides ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Essential Components ◽  
Exclusion Chromatography

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1–RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1–RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1–RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1–RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1–RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1–RuvBL2 complex does not fit the recent structural model of the yeast Rvb1–Rvb2 complex.

scholarly journals Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alexander Moysa ◽  
Dietmar Hammerschmid ◽  
Roman H. Szczepanowski ◽  
Frank Sobott ◽  
Michal Dadlez
Keyword(s):  
Analytical Ultracentrifugation ◽  
Vascular Diseases ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Size Exclusion ◽  
Glycation End Products ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

AbstractThe pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen–deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

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.

Characterization of the novelTrypanosoma bruceiinosine 5′-monophosphate dehydrogenase

Parasitology ◽  
2013 ◽  
Vol 140 (6) ◽  
pp. 735-745 ◽  
Author(s):  
TOMOAKI BESSHO ◽  
SHOKO MORII ◽  
TOSHIHIDE KUSUMOTO ◽  
TAKAHIRO SHINOHARA ◽  
MASANORI NODA ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
De Novo ◽  
Binding Mode ◽  
Nucleotide Metabolism ◽  
Sub Saharan Africa ◽  
Size Exclusion ◽  
Purine Nucleotides ◽  
Exclusion Chromatography ◽  
Sub Saharan ◽  
Monophosphate Dehydrogenase

SUMMARYThere is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy.Trypanosomalacks the enzymatic machinery for thede novosynthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5′-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinantTrypanosoma bruceiIMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculatedKmvalues of 30 and 1300 μmfor IMP and NAD, respectively. The obtainedKmvalue of TbIMPDH for NAD is approximately 20–200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies showKivalues of 3·2 μm, 21 nM and 3·3 nM for ribavirin 5′-monophosphate, mycophenolic acid and mizoribine 5′-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

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