scholarly journals Sedimentation velocity analysis of TMPyP4-induced dimer formation of human telomeric G-quadruplex

RSC Advances ◽  
2017 ◽  
Vol 7 (87) ◽  
pp. 55098-55105 ◽  
Author(s):  
Yating Gao ◽  
Tianlei Guang ◽  
Xiaodong Ye
Keyword(s):  
Analytical Ultracentrifugation ◽  
Sedimentation Velocity ◽  
Velocity Analysis ◽  
Dimer Formation ◽  
G Quadruplex

Analytical ultracentrifugation sedimentation velocity (AUC-SV) was used to study the interactions between TMPyP4 and AGGG(TTAGGG)3 (Tel22) and the TMPyP4-induced dimer formation of G-quadruplex.

scholarly journals A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

2021 ◽  
Vol 11 ◽  
Author(s):  
Orla M. Dunne ◽  
Xin Gao ◽  
Ruodan Nan ◽  
Jayesh Gor ◽  
Penelope J. Adamson ◽  
...  
Keyword(s):  
Host Cell ◽  
Analytical Ultracentrifugation ◽  
Dissociation Constants ◽  
Alternative Pathway ◽  
Factor H ◽  
Regulatory Control ◽  
Size Exclusion ◽  
Complement Factor ◽  
Dimer Formation ◽  
Self Association

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

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

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.

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