Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling

Schuck, Peter

doi:10.1016/s0006-3495(00)76713-0

Cited by 3,593 publications

(3,514 citation statements)

References 53 publications

Supporting

Mentioning

3,468

Contrasting

Unclassified

Order By: Relevance

“…Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c ( s ) analysis in SEDFIT (Schuck, 2000), and the results of this fit are shown (solid lines, rainbow color scheme). (B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown.…”

Section: Resultsmentioning

confidence: 99%

“…The FDS‐SV data were sorted according to the sample position, PMT voltage, and gains using the Fluorescence Tools set in SEDFIT version 14.4 (Zhao et al , 2013b), and subjected to standard c ( s ) analysis (Schuck, 2000) in SEDFIT. Integration between 1 and 6 S was carried out to generate the isotherm analysis of signal‐weighted sedimentation coefficients, s w .…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

Self Cite

View full text Add to dashboard Cite

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…Protein concentration distribution was monitored at 280 nm, at 235,000 g and 20°C. Time‐derivative analysis was computed using the SEDFIT software package, version 12.1b (Schuck, 2000), resulting in a c(s) distribution and an estimate for the molecular weight Mf (from the sedimentation coefficient and the diffusion coefficient, as inferred from the broadening of the sedimentation boundary, assuming all observed species share the same frictional coefficient f/f0).…”

Section: Methodsmentioning

confidence: 99%

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

et al. 2016

View full text Add to dashboard Cite

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

show abstract

“…This concentration range is comparable to the plasma concentration of C4 which is 0.24 mg/ml, while being less than that of 1.15 mg/ml for C3 [40]. The peaks in the size-distribution analyses c(s) monitor the extent of sample monodispersity, and the shape is monitored through the sedimentation coefficient s 20,w of the observed peaks [21]. Data analyses resulted in good boundary fits for C4b using both absorbance and interference optics (left panels, Figure 5).…”

Section: Auc Analyses Of C4b In Solutionmentioning

confidence: 71%

“…The buffer density was measured using an Anton-Paar DMA 5000 density meter to be 1.00487 and 1.00195 g/ml for the 137 and 50 mM NaCl buffers, respectively. SEDFIT version 14.6 was used to fit the experimental boundaries directly according to the Lamm equation [21]. Size-distribution analyses c(s) were produced by SEDFIT that assumed that all species have the same frictional ratio f/f o .…”

Section: Auc Measurements Of C4bmentioning

confidence: 99%

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

Fung

Wright

Gor

et al. 2016

Biochemical Journal

View full text Add to dashboard Cite

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg 104 -Glu 1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s 20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar R G values of 4.89-4.90 nm for C4b in 137-250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137-250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED-MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b.

show abstract

Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling

Cited by 3,593 publications

References 53 publications

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

Contact Info

Product

Resources

About