Probing RNA–Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation

2020 ◽  
pp. 281-317 ◽  
Author(s):  
Somdeb Mitra ◽  
Borries Demeler
Keyword(s):  
Protein Interactions ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Velocity

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

2011 ◽  
Vol 436 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Masanori Noda ◽  
Susumu Uchiyama ◽  
Adam R. McKay ◽  
Akihiro Morimoto ◽  
Shigeki Misawa ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Analytical Ultracentrifugation ◽  
Three Dimensional ◽  
Sedimentation Velocity ◽  
Dimensional Structure ◽  
Histone H2a ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A–H2B dimer or H3–H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein–protein interactions in solution.

scholarly journals Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Huaying Zhao ◽  
Yan Fu ◽  
Carla Glasser ◽  
Eric J Andrade Alba ◽  
Mark L Mayer ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ampa Receptors ◽  
Cell Biology ◽  
Analytical Ultracentrifugation ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Sedimentation Velocity ◽  
Excitation Wavelength ◽  
Amino Terminal ◽  
High Affinity

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors.

Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): Limits of quantitation for a monoclonal antibody system

2007 ◽  
Vol 361 (1) ◽  
pp. 24-30 ◽  
Author(s):  
John P. Gabrielson ◽  
Theodore W. Randolph ◽  
Brent S. Kendrick ◽  
Michael R. Stoner
Keyword(s):  
Monoclonal Antibody ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Velocity
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