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 Immunoassay for Sex Hormone-Binding Globulin in Undiluted Serum Is Influenced by High-Molecular-Mass Aggregates

2005 ◽  
Vol 51 (2) ◽  
pp. 401-407 ◽  
Author(s):  
Markus Thaler ◽  
Jochen Metzger ◽  
Anita Schreiegg ◽  
Barbara Denk ◽  
Andreas Gleixner ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
High Molecular Mass ◽  
Sex Hormone ◽  
Size Exclusion ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Spr Biosensor ◽  
Undiluted Serum ◽  
Exclusion Chromatography

Abstract Background: The new Elecsys® chemiluminescence assay for measurement of homodimeric sex hormone-binding globulin (SHBG) was designed for use with undiluted serum, in contrast to other methods that require predilution. During assay development, unexpected calibration difficulties were observed that were attributable to particular biochemical properties of the highly concentrated SHBG in solution. Methods: We used a surface plasmon resonance (SPR) biosensor, which enables biomolecular interaction analysis of SHBG, and size-exclusion chromatography for this investigation. The immunoassay was evaluated for imprecision, linearity, and suitability of the dilution medium, and the method was compared with an IRMA for SHBG. Results: The SPR biosensor characterized the special protein properties of SHBG in various concentrations. Above 200 nmol/L there was a strong tendency toward formation of high-molecular-mass aggregates. This was also detectable by size-exclusion chromatography and could be reversed by simple dilution of the sample. On the basis of these results, the dynamic measuring range of the SHBG assay is restricted to 0.350–200 nmol/L. Assay evaluation on a 2010 analyzer revealed excellent precision (CV ≤2.5%). Mean recoveries were 84.2–98.8%. Intermethod comparison with an IRMA yielded a satisfactory concordance of the two assays with a Spearman correlation coefficient of 0.8807. Conclusions: Aggregates of human SHBG may have a detrimental impact on the accurate measurement of the protein if measurements are performed with undiluted serum samples. Further work is needed to clarify whether these high-molecular-mass aggregates influence the free fraction of steroid hormones in vivo.

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 Crystallization and preliminary X-ray characterization of the full-length bacteriophytochrome from the plant pathogenXanthomonas campestrispv.campestris

2014 ◽  
Vol 70 (12) ◽  
pp. 1636-1639 ◽  
Author(s):  
Sebastián Klinke ◽  
Lisandro H. Otero ◽  
Jimena Rinaldi ◽  
Santiago Sosa ◽  
Beatriz G. Guimarães ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Data Bank ◽  
Full Length ◽  
Size Exclusion ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Exclusion Chromatography ◽  
Output Module

Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2–GAF–PHY domains) and a C-terminal variable output module. The bacteriumXanthomonas campestrispv.campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified to homogeneity by nickel–NTA affinity and size-exclusion chromatography and was then crystallized at room temperature bound to its cofactor biliverdin. A complete native X-ray diffraction data set was collected to a maximum resolution of 3.25 Å. The crystals belonged to space groupP43212, with unit-cell parametersa=b= 103.94,c= 344.57 Å and a dimer in the asymmetric unit. Refinement is underway after solving the structure by molecular replacement.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (10) ◽  
pp. 1362-1367 ◽  
Author(s):  
Emmanuel Nji ◽  
Dianfan Li ◽  
Declan A. Doyle ◽  
Martin Caffrey
Keyword(s):  
Metal Ion ◽  
Cell Volume Regulation ◽  
Ion Concentration ◽  
Size Exclusion ◽  
Substrate Selectivity ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

scholarly journals PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ho-Ryun Chung ◽  
Chao Xu ◽  
Alisa Fuchs ◽  
Andreas Mund ◽  
Martin Lange ◽  
...  
Keyword(s):  
Dna Damage Response ◽  
Size Exclusion ◽  
X Ray ◽  
Chip Sequencing ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Damage Response ◽  
Exclusion Chromatography ◽  
Insight Into ◽  
Binding Cell

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

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