Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii

2012 ◽  
Vol 444 (2) ◽  
pp. 219-226 ◽  
Author(s):  
Cong Han ◽  
Gareth S. A. Wright ◽  
Karl Fisher ◽  
Stephen E. J. Rigby ◽  
Robert R. Eady ◽  
...  
Keyword(s):  
Sequence Data ◽  
Gel Filtration ◽  
Particle Diameter ◽  
Size Exclusion ◽  
Gyration Radius ◽  
Saxs Data ◽  
Ralstonia Pickettii ◽  
Exclusion Chromatography ◽  
Data Yield

NiRs (nitrite reductases) convert nitrite into NO in the denitrification process. RpNiR (Ralstonia pickettii NiR), a new type of dissimilatory Cu-containing NiR with a C-terminal haem c domain from R. pickettii, has been cloned, overexpressed in Escherichia coli and purified to homogeneity. The enzyme has a subunit molecular mass of 50515 Da, consistent with sequence data showing homology to the well-studied two-domain Cu NiRs, but with an attached C-terminal haem c domain. Gel filtration and combined SEC (size-exclusion chromatography)-SAXS (small angle X-ray scattering) analysis shows the protein to be trimeric. The metal content of RpNiR is consistent with each monomer having a single haem c group and the two Cu sites being metallated by Cu2+ ions. The absorption spectrum of the oxidized as-isolated recombinant enzyme is dominated by the haem c. X-band EPR spectra have clear features arising from both type 1 Cu and type 2 Cu centres in addition to those of low-spin ferric haem. The requirements for activity and low apparent Km for nitrite are similar to other CuNiRs (Cu-centre NiRs). However, EPR and direct binding measurements of nitrite show that oxidized RpNiR binds nitrite very weakly, suggesting that substrate binds to the reduced type 2 Cu site during turnover. Analysis of SEC-SAXS data suggests that the haem c domains in RpNiR form extensions into the solvent, conferring a high degree of conformational flexibility in solution. SAXS data yield Rg (gyration radius) and Dmax (maximum particle diameter) values of 43.4 Å (1 Å=0.1 nm) and 154 Å compared with 28 Å and 80 Å found for the two-domain CuNiR of Alcaligenes xylosoxidans.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

LUTI: a double-function inhibitor isolated from naked flax seeds

2019 ◽  
Vol 51 (10) ◽  
pp. 989-996
Author(s):  
Lei Wang ◽  
Yinglu Chen ◽  
Feng Wu ◽  
Shasha Wu ◽  
Xiaojun Hu ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Lactic Acid Production ◽  
Gel Filtration ◽  
Glucose Consumption ◽  
Competitive Inhibitor ◽  
Size Exclusion ◽  
Kinetic Experiment ◽  
Glucosidase Inhibitors ◽  
Exclusion Chromatography ◽  
Inhibitor Type

Abstract Acute glucose fluctuation during the postprandial period causes a risk for type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors have been approved as therapeutic agents for diabetes. In the present study, a protein with α-glucosidase inhibitory activity from Flax (Linum usitatissimum) seeds was isolated using a one-step purification with Q-Sepharose4B column, followed by Sephacryl S-200 size-exclusion chromatography. It was identified as a trypsin inhibitor, named L. usitatissimum trypsin inhibitor (LUTI). The half maximal inhibitory concentration (IC50) of LUTI was 113.92 μM for α-glucosidase and 6.17 μM for trypsin. Lineweaver–Burk kinetic experiment showed that the protein exhibited two distinct inhibitory modes, a competitive inhibitor type for α-glucosidase and a non-competitive type for trypsin. The interaction between LUTI and α-glucosidase was detected through gel filtration chromatography and dynamic light scattering. Increased glucose consumption and lactic acid production were also observed following LUTI treatment in Caco-2 and HepG2 cells. LUTI inhibits not only the activity of trypsin but also the activity of α-glucosidase. It is expected that LUTI will become an oral hypoglycemic polypeptide drug for T2DM.

Introducing SEC–SANS for studies of complex self-organized biological systems

2018 ◽  
Vol 74 (12) ◽  
pp. 1178-1191 ◽  
Author(s):  
Nicolai Tidemand Johansen ◽  
Martin Cramer Pedersen ◽  
Lionel Porcar ◽  
Anne Martel ◽  
Lise Arleth
Keyword(s):  
Small Angle ◽  
Signal To Noise Ratio ◽  
Biological Systems ◽  
Size Exclusion ◽  
Contrast Variation ◽  
Saxs Data ◽  
Biological Structures ◽  
Exclusion Chromatography ◽  
Protein Components ◽  
Counting Statistics

Small-angle neutron scattering (SANS) is maturing as a method for studying complex biological structures. Owing to the intrinsic ability of the technique to discern between 1H- and 2H-labelled particles, it is especially useful for contrast-variation studies of biological systems containing multiple components. SANS is complementary to small-angle X-ray scattering (SAXS), in which similar contrast variation is not easily performed but in which data with superior counting statistics are more easily obtained. Obtaining small-angle scattering (SAS) data on monodisperse complex biological structures is often challenging owing to sample degradation and/or aggregation. This problem is enhanced in the D2O-based buffers that are typically used in SANS. In SAXS, such problems are solved using an online size-exclusion chromatography (SEC) setup. In the present work, the feasibility of SEC–SANS was investigated using a series of complex and difficult samples of membrane proteins embedded in nanodisc particles that consist of both phospholipid and protein components. It is demonstrated that SEC–SANS provides data of sufficient signal-to-noise ratio for these systems, while at the same time circumventing aggregation. By combining SEC–SANS and SEC–SAXS data, an optimized basis for refining structural models of the investigated structures is obtained.

scholarly journals US-SOMO HPLC-SAXS module: dealing with capillary fouling and extraction of pure component patterns from poorly resolved SEC-SAXS data

2016 ◽  
Vol 49 (5) ◽  
pp. 1827-1841 ◽  
Author(s):  
Emre Brookes ◽  
Patrice Vachette ◽  
Mattia Rocco ◽  
Javier Pérez
Keyword(s):  
High Performance ◽  
Pure Component ◽  
Size Exclusion ◽  
Baseline Correction ◽  
Saxs Data ◽  
X Ray ◽  
Exclusion Chromatography ◽  
Flow Through ◽  
Gaussian Decomposition ◽  
Comprehensive Framework

Size-exclusion chromatography coupled with SAXS (small-angle X-ray scattering), often performed using a flow-through capillary, should allow direct collection of monodisperse sample data. However, capillary fouling issues and non-baseline-resolved peaks can hamper its efficacy. The UltraScan solution modeler (US-SOMO) HPLC-SAXS (high-performance liquid chromatography coupled with SAXS) module provides a comprehensive framework to analyze such data, starting with a simple linear baseline correction and symmetrical Gaussian decomposition tools [Brookes, Pérez, Cardinali, Profumo, Vachette & Rocco (2013). J. Appl. Cryst. 46, 1823–1833]. In addition to several new features, substantial improvements to both routines have now been implemented, comprising the evaluation of outcomes by advanced statistical tools. The novel integral baseline-correction procedure is based on the more sound assumption that the effect of capillary fouling on scattering increases monotonically with the intensity scattered by the material within the X-ray beam. Overlapping peaks, often skewed because of sample interaction with the column matrix, can now be accurately decomposed using non-symmetrical modified Gaussian functions. As an example, the case of a polydisperse solution of aldolase is analyzed: from heavily convoluted peaks, individual SAXS profiles of tetramers, octamers and dodecamers are extracted and reliably modeled.

scholarly journals Characterization of the oligomeric states of the CK2 α2β2 holoenzyme in solution

2017 ◽  
Vol 474 (14) ◽  
pp. 2405-2416 ◽  
Author(s):  
Graziano Lolli ◽  
Denise Naressi ◽  
Stefania Sarno ◽  
Roberto Battistutta
Keyword(s):  
Active Form ◽  
Mass Spectrometry Data ◽  
Size Exclusion ◽  
Saxs Data ◽  
Deconvolution Analysis ◽  
Exclusion Chromatography ◽  
Kinase Ck2 ◽  
First Time

The regulatory mechanism of protein kinase CK2 has still to be fully clarified. The prevailing hypothesis is that CK2 is controlled by a self-polymerisation mechanism leading to inactive supramolecular assemblies that, when needed, can be disassembled into the α2β2 monomer, the active form of the holoenzyme. In vitro, monomeric α2β2 seems present only at high ionic strengths, typically 0.35–0.50 M NaCl, while at lower salt concentrations oligomers are formed. In the present study, size-exclusion chromatography (SEC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS) and mutagenesis have been employed for the characterization of the oligomeric states of CK2 in solution. SAXS measurements at 0.35 M NaCl show for the first time the shape of the α2β2 active monomer in solution. At 0.25 M salt, despite single average properties indicating an aggregated holoenzyme, deconvolution analysis of SAXS data reveals an equilibrium involving not only circular trimeric and linear oligomeric (3–4 units) forms of α2β2, but also considerable amounts of the monomer. Together SAXS and mutagenesis confirm the presence in solution of the oligomers deduced by crystal structures. The lack of intermediate species such as αβ2, α or β2 indicates that the holoenzyme is a strong complex that does not spontaneously dissociate, challenging what was recently proposed on the basis of mass spectrometry data. A significant novel finding is that a considerable amount of monomer, the active form of CK2, is present also at low salt. The solution properties of CK2 shown in the present study complement the model of regulation by polymerization.

Distinct Instrumental Approaches to SAXS

2018 ◽  
Author(s):  
Eaton E. Lattman ◽  
Thomas D. Grant ◽  
Edward H. Snell
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Size Exclusion ◽  
Saxs Data ◽  
Sample Handling ◽  
X Ray ◽  
Macromolecular Conformation ◽  
Exclusion Chromatography ◽  
Membrane Bound ◽  
Instrumental Approaches

There are more specialized applications of SAXS and SANS which require specific experimental considerations. This chapter covers size exclusion chromatography which has proven to be useful to study both soluble and membrane bound proteins allowing the study of samples that show time and concentration dependent dynamics. It also describes iime-resolved techniques for SAXS and in a few cases, SANS. Finally, with improved X-ray sources, detectors, sample handling, and compute power, the ability to perform SAXS data in high-throughput is available. This is discussed in enabling the use of SAXS to study protein interactions, map macromolecular conformation, and rapidly characterize samples amongst other applications.

scholarly journals Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme

1993 ◽  
Vol 292 (3) ◽  
pp. 797-803 ◽  
Author(s):  
E Mertens ◽  
J De Jonckheere ◽  
E Van Schaftingen
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Potassium Thiocyanate ◽  
Enzyme Concentration ◽  
Size Exclusion ◽  
Maximal Effect ◽  
Naegleria Fowleri ◽  
Exclusion Chromatography ◽  
Nægleria Fowleri ◽  
Fructose 1,6 Bisphosphate

PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.

Crystal and solution structures of fragments of the human leucocyte common antigen-related protein

2020 ◽  
Vol 76 (5) ◽  
pp. 406-417
Author(s):  
Joachim Vilstrup ◽  
Amanda Simonsen ◽  
Thea Birkefeldt ◽  
Dorthe Strandbygård ◽  
Jeppe Lyngsø ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Size Exclusion ◽  
Receptor Protein ◽  
Type I ◽  
Common Antigen ◽  
Heparin Binding ◽  
Related Protein ◽  
Saxs Data ◽  
Exclusion Chromatography ◽  
The Individual

Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1–4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1–4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1–3) and the four C-terminal FNIII domains (LARFN5–8) both bound heparin, while LARFN1–4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1–3 and LARFN5–8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.

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