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.

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.

scholarly journals Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

2005 ◽  
Vol 71 (9) ◽  
pp. 5501-5510 ◽  
Author(s):  
Sandra W. A. Hinz ◽  
Marieke I. Pastink ◽  
Lambertus A. M. van den Broek ◽  
Jean-Paul Vincken ◽  
Alphons G. J. Voragen
Keyword(s):  
Molecular Mass ◽  
Transmembrane Domain ◽  
Bifidobacterium Longum ◽  
Cell Extract ◽  
Size Exclusion ◽  
Glycoside Hydrolase Family ◽  
Type I ◽  
Native Molecular Mass ◽  
Exclusion Chromatography ◽  
Hydrolase Family

ABSTRACT A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and V max for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

scholarly journals Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing

2020 ◽  
Vol 76 (9) ◽  
pp. 899-904
Author(s):  
Ke Shi ◽  
Fredy Kurniawan ◽  
Surajit Banerjee ◽  
Nicholas H. Moeller ◽  
Hideki Aihara
Keyword(s):  
Crystal Structure ◽  
Bacteriophage T4 ◽  
Early Gene ◽  
Size Exclusion ◽  
Amino Terminal ◽  
Intramolecular Disulfide Bond ◽  
Exclusion Chromatography ◽  
T4 Bacteriophage ◽  
Sad Phasing ◽  
T4 Phage

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

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.

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 Crystal structure of an anti-idiotype variable lymphocyte receptor

2017 ◽  
Vol 73 (12) ◽  
pp. 682-687
Author(s):  
Bernard C. Collins ◽  
Hiro Nakahara ◽  
Sharmistha Acharya ◽  
Max D. Cooper ◽  
Brantley R. Herrin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Similarity ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Size Exclusion ◽  
Antigen Receptors ◽  
Diagnostic Assays ◽  
Great Utility ◽  
Exclusion Chromatography ◽  
Leukemic Clone

Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39–BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.

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 Type III CRISPR complexes from Thermus thermophilus.

2016 ◽  
Vol 63 (2) ◽  
Author(s):  
Marta Szychowska ◽  
Wojciech Siwek ◽  
Damian Pawolski ◽  
Asgar Abbas Kazrani ◽  
Krzysztof Pyrc ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Mass Spectroscopy ◽  
Thermus Thermophilus ◽  
Size Exclusion ◽  
Acquired Immunity ◽  
Type I ◽  
Type Iii ◽  
Exclusion Chromatography

Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.

Sign in / Sign up

Export Citation Format

Share Document