Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Brennich, Martha; Round, Adam; Hutin, Stephanie

doi:10.3791/54861

Cited by 32 publications

(28 citation statements)

References 22 publications

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“…As a concentration dependent increase in size was detectable further analysis were based solely on the data collected at 1 mg/ml. In a similar manner as described above, TbMORN1(2-15) data were collected at ESRF BM29 beamline (Pernot, Round et al, 2013) in SEC-SAXS mode with the setup described in by Brennich et al (Brennich, Round et al, 2017). SAXS data from the run were collected at a wavelength of 0.99 Å using a sample-to-detector (PILATUS 1 M, DECTRIS) distance of 2.867 m. Here too, a Superdex 200 10/300 (GE Healthcare) column was used as well as 20 mM Tris-HCl pH 7.5, 100 mM NaCl as mobile phase.…”

Section: Methodsmentioning

confidence: 99%

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

Sajko

Grishkovskaya

Košťan

et al. 2019

Preprint

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38MORN (membrane occupation and recognition nexus) repeat proteins have a wide taxonomic 39 distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain 40 poorly characterised at both a structural and a functional level compared to other common 41 repeat motifs such as leucine-rich repeats, armadillo repeats, WD40 repeats, and ankyrin 42 repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate 43 membrane targeting, but direct evidence for this role is actually lacking. This putative activity 44 was addressed by focusing on a protein composed solely of MORN repeats -Trypanosoma 45 brucei MORN1. No evidence for binding to membranes or lipid vesicles by TbMORN1 either 46 in vivo or in vitro could be obtained. TbMORN1 did interact with individual phospholipids, but 47 it remains unclear if this was physiological or an artefact. High-and low-resolution structures 48 of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites 49 Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of 50 macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. The 51 structures indicated that MORN repeats can mediate homotypic interactions, and can function 52 as both dimerisation and oligomerisation devices.53 54 93 al., 2006, Im, Davis et al., 2007, Camacho, Smertenko et al., 2009). It therefore remains 94 unclear what role(s) this ubiquitous class of repeat motifs actually have (Mikami, Saavedra et 95 al., 2010). 97Coupled to this lack of unambiguous functional data is a lack of high-resolution structural 98 information, exemplified by the ongoing lack of consensus as to whether a single repeat is 14 99 4 or 23 amino acids. This contrasts sharply with the considerable amount of information 100 available on other classes of protein repeat motifs such as ankyrin repeats, leucine-rich 101 repeats, or WD40 repeats (Andrade, Perez-Iratxeta et al., 2001). Until very recently, the 102 structure of the SETD7 histone methyltransferase was the sole representative of the MORN 103 repeat protein family in the protein data bank (PDB) (Jacobs, Harp et al., 2002, Wilson et al., 104 2002, Xiao, Jing et al., 2003. Even here, the structure of the N-terminal domain containing 105 the repeats is incomplete, and the level of sequence similarity of the repeats to those of 106 junctophilins and other family members makes assignment difficult. Each repeat appears to 107 form a β-hairpin with an acidic surface, but it remains unclear if this is a general property of 108 MORN repeats. The SETD7 structure has not been analysed in this context, with more work 109 focusing on its catalytic methyltransferase domain. In 2019, and while this manuscript was in 110 preparation, Li et al. published the first high-resolution structure of a canonical MORN repeat 111 protein, specifically the MORN4/retinophilin protein in complex with its Myo3a binding partner 112 (Li, Liu et al., 2019). This structure contains ...

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Section: Methodsmentioning

confidence: 99%

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

Sajko

Grishkovskaya

Košťan

et al. 2019

Preprint

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“…SAXS experiments on ODA16 or the ODA16-IFT46N complex were performed at the BM29 Beamline (European Synchrotron Radiation Facility, Grenoble, France) using a Pilatus 1M detector using a similar protocol the one used previously (53). In brief, SAXS data were collected on proteins and protein complexes eluting directly from a Superdex 200 10/300 GL SEC column.…”

Section: Small-angle X-ray Scattering Of Oda16 and Oda16-ift46nmentioning

confidence: 99%

Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46

Täschner

Mourão

Awasthi

et al. 2017

Journal of Biological Chemistry

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Motile cilia are found on unicellular organisms such as the green alga , on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed β-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of , that the C-terminal β-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

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“…We applied the SEC-SAXS method that allowed us to collect and interpret SAXS data on experimentally difficult aggregation-prone protein system, such as truncated recPrP, and to minimize radiation damage thanks to the continuous flow (28). To dissect the different conformational recPrP states we used EOM approaches (37–38) that provided a semi-quantitative assessment on the structural effect of Cu(II) to protein compactness through direct comparison of the size distributions of the apo versus Cu(II)-conformers.…”

Section: Discussionmentioning

confidence: 99%

“…Given the sensitivity for batch SAXS mode for even small amounts of large soluble aggregates we used SEC-SAXS approaches to measure SAXS data only on monodisperse samples. A volume of 250 μL of protein per each BvPrP sample ( apo and copper-loaded) at 12 mg/mL was loaded on a GE Superdex 75 10/300 GL column, and a volume of 50 μL protein per each OvPrP ARR and OvPrP VRQ ( apo and copper-loaded) samples at 7 mg/mL was loaded on a GE Superdex 200 5/150 GL column via a high performance liquid chromatography (HPLC) system (DGU-20A5R, Shimadzu, France) attached directly to the sample-inlet valve of the BM29 sample changer (28). All the apo samples were measured in buffer 25 mM Sodium Acetate, 250 mM NaCl, pH 5.5 at 20 °C.…”

Section: Methodsmentioning

confidence: 99%

Deciphering copper coordination in the animal prion protein amyloidogenic domain

Salzano

Brennich

Mancini

et al. 2018

Preprint

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18Prions are pathological isoforms of the cellular prion protein (PrP C ) responsible for transmissible 19 spongiform encephalopathies (TSE). PrP C interacts with copper through unique octarepeat and 20 non-octarepeat (non-OR) binding sites. Previous works on human PrP C suggest that copper 21 binding to the non-OR region may have a role during prion conversion. The molecular details of 22 copper coordination within the non-OR region are not well characterized. By means of small 23 angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) 24 spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its 25 coordination geometries when bound to the non-OR region of recombinant PrP C (recPrP) from 26 animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine 27 PrP C carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); 28 while as highly TSE-susceptible PrP C models we employed OvPrP with polymorphism V136, 29R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) 30 affects the structural plasticity of the non-OR region leading to a more compacted conformation 31 of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal 32 species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues 33 (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and 34 BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water 35 molecule, making the non-OR region more flexible and open to the solvent. These changes in 36 copper coordination in prion resistant and susceptible species provide new insights into the 37 molecular mechanisms governing the resistance or susceptibility of certain species to TSE. Keywords 42 Prion protein, SAXS, Ensemble Optimization Method, EXAFS spectroscopy, non-OR region, 43 copper coordination, transmissible spongiform encephalopathies.44 45 48 of the physiological cellular prion protein (PrP C ) into a β-sheet rich isoform called prion or PrP Sc 49(1). TSE are rare disorders that can be sporadic, genetic or infectious. Animal TSE include 50 scrapie in sheep and goats, chronic wasting disease (CWD) in cervids and bovine spongiform 51 encephalopathy (BSE) in cattle (2). 52The PrP C structure consists in a C-terminal folded domain (from residues 128 to 231, hereafter in 53 human numbering) mainly containing α-helical motifs and two short anti-parallel β-sheets (3). 54On the contrary, the N-terminal moiety (residues 23-127) is largely unstructured (4) and features 55 an octapeptide-repeat region (OR) (residues 61-91) composed by four octapeptides, each 56 carrying histidines able to coordinate prevalently one copper ion, Cu(II) (5). Cu(II) can also bind 57 at two additional histidines -H96 and H111 with coordination from the imidazole rings and 58 nearby backbone amides-located in a segment (residues 90-111) called "fifth" or n...

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Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Cited by 32 publications

References 22 publications

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46

Deciphering copper coordination in the animal prion protein amyloidogenic domain

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