Analytical Ultracentrifugation Data Analysis with UltraScan-III

Demeler, Borries; Gorbet, Gary E.

doi:10.1007/978-4-431-55985-6_8

Cited by 103 publications

(117 citation statements)

References 22 publications

Supporting

Mentioning

114

Contrasting

Unclassified

Order By: Relevance

“…The scans also were analyzed by the time‐derivative method to obtain the sedimentation coefficient distribution, g(s*) (Stafford, ). All sedimentation coefficients are expressed in Svedberg units (S); one Svedberg is equal to 10 −13 s. All data editing and analyses were conducted using the UltraScanIII software (Demeler & Gorbet, in press).…”

Section: Methodsmentioning

confidence: 99%

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

et al. 2016

View full text Add to dashboard Cite

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg2+‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in the absence of MgCl2, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.

show abstract

Section: Methodsmentioning

confidence: 99%

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Lysozyme, for use as a scattering standard, was solubilized in 150 mM NaCl, 40 mM sodium acetate, pH 3.8, and dialyzed against the same buffer with the final dialysate 22 for details on intensity measurements, which allow up to two samples to be measured per cell). All data were analyzed using UltraScan-III, revision 2029 (23). Time and radially invariant noise was removed during analysis with the two-dimensional spectrum analysis (24), and diffusion-corrected sedimentation coefficient distributions were calculated with the enhanced van HoldeWeischet method (25).…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1

Rubio-Marrero

Vincelli

Jeffries

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and biolayer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system. Contactin-associated protein-like 2 (CASPR2)6 is a neuronal cell adhesion molecule known in rodents to be necessary for the clustering of the Kv1 potassium channels at juxtaparanodes (1). In myelinated nerves, CASPR2 is confined to the juxtaparanodal region of the axon where it appears to associate with the immunoglobulin domains of TAG-1 (transient axonal glycoprotein-1) to form a scaffold, which clusters the potassium channels Kv1.1 and Kv1.2 (2-4).CASPR2 is predicted to be a type I transmembrane protein of 1331 amino acids with the extracellular domain followed by a single transmembrane domain and a short (48 residues) intracellular domain that terminates with a class II PDZ recognition motif. Computational predictions suggest that CASPR2 has 12 putative N-linked glycosylation sites and 36 Cys residues likely making 18 disulfide bonds, forming 8 independently folded domains: four laminin, neurexin, sex hormone-binding globulin domains (LNS), two epidermal growth factor (EGF) domains, one discoidin domain, and one fibrinogen-like domain (Fig. 1A). CASPR2 shares an overall domain organization with ␣-neurexin-1 despite a relatively low amino acid identity (ϳ23% identity, ϳ39% similarity). However, distinctive features such as a discoidin domain in place of the first LNS domain and a fibrinogen-like domain in place of the 4th LNS domain suggest a different overall structural architecture. No information about the three-dimensional structure of CASPR2, other than that inferred from sequence homology, is currently available. Functionally, only TAG-1 (contactin 2 or CNTN2) has been thus far identified as the extracellular ligand for CASPR2 (2-4).Individuals in a cohort of Amish children, homozygous for a frameshift mutation (single-base G deletion at nucleotide 3709 in exon 22) involving the CNTNAP2 gene, ...

show abstract

“…These experiments are more effective, although limited to extremely monodisperse systems, for determination of molecular weight ( M w ) of proteins as well as investigation of protein–protein interactions2122. Data analysis software available such as Ultrascan23 or SEDFIT1724 provide user-friendly platforms to assess the quality of data and pre-defined models for homogeneous/heterogeneous protein–protein interactions with different stoichiometry. Most of the models, however, require well-defined molecular weight ( M w ) and extinction coefficients ( ɛ ) of the interacting species.…”

mentioning

confidence: 99%

A centrifugation-based physicochemical characterization method for the interaction between proteins and nanoparticles

Bekdemir

Stellacci

2016

Nat Commun

View full text Add to dashboard Cite

Nanomedicine requires in-depth knowledge of nanoparticle–protein interactions. These interactions are studied with methods limited to large or fluorescently labelled nanoparticles as they rely on scattering or fluorescence-correlation signals. Here, we have developed a method based on analytical ultracentrifugation (AUC) as an absorbance-based, label-free tool to determine dissociation constants (KD), stoichiometry (Nmax), and Hill coefficient (n), for the association of bovine serum albumin (BSA) with gold nanoparticles. Absorption at 520 nm in AUC renders the measurements insensitive to unbound and aggregated proteins. Measurements remain accurate and do not become more challenging for small (sub-10 nm) nanoparticles. In AUC, frictional ratio analysis allows for the qualitative assessment of the shape of the analyte. Data suggests that small-nanoparticles/protein complexes significantly deviate from a spherical shape even at maximum coverage. We believe that this method could become one of the established approaches for the characterization of the interaction of (small) nanoparticles with proteins.

show abstract

Analytical Ultracentrifugation Data Analysis with UltraScan-III

Cited by 103 publications

References 22 publications

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1

A centrifugation-based physicochemical characterization method for the interaction between proteins and nanoparticles

Contact Info

Product

Resources

About