Analytical Ultracentrifugation as a Matrix-Free Probe for the Study of Kinase Related Cellular and Bacterial Membrane Proteins and Glycans

Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free—providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review—designed for the non-hydrodynamic expert—examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system—studied in aqueous and detergent based solvent systems—and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.

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Insight into protein–protein interactions from analytical ultracentrifugation

Biochemical Society Transactions ◽

10.1042/bst0380901 ◽

2010 ◽

Vol 38 (4) ◽

pp. 901-907 ◽

Cited By ~ 37

Author(s):

Stephen E. Harding ◽

Arthur J. Rowe

Keyword(s):

Protein Interactions ◽

Dynamic Range ◽

Analytical Ultracentrifugation ◽

Dissociation Constants ◽

Interaction Strength ◽

Sedimentation Equilibrium ◽

Solution Technique ◽

Protein Protein Interactions ◽

Self Association ◽

Made In

Analytical ultracentrifugation is a free solution technique with no supplementary immobilization, columns or membranes required, and can be used to study self-association and hetero-interactions, stoichiometry, reversibility and interaction strength across a very large dynamic range (dissociation constants from 10−12 M to 10−1 M). In the present paper, we review some of the advances that have been made in the two different types of sedimentation experiment – sedimentation equilibrium and sedimentation velocity – for the analysis of protein–protein interactions and indicate how major complications such as thermodynamic and hydrodynamic non-ideality can be dealt with.

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Correlated STORM-homoFRET Imaging: Applications to the Study of Membrane Receptor Self-Association and Clustering

10.1101/2021.12.21.473581 ◽

2021 ◽

Author(s):

Amine Driouchi ◽

Scott Gray-Owen ◽

Christopher M Yip

Keyword(s):

Spatial Distribution ◽

Membrane Proteins ◽

Complex Mixture ◽

Cell Surface Receptor ◽

Oligomeric State ◽

Imaging Approach ◽

Surface Receptor ◽

A Cell ◽

Self Association ◽

And Function

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. We report here on a correlated STORM/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Live cell homoFRET imaging of CEACAM1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, revealed highly heterogenous diffraction-limited structures on the surface of HeLa cells. Correlated super-resolved STORM imaging revealed that these structures comprised a complex mixture and spatial distribution of self-associated CEACAM1 molecules. This correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

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

Frontiers in Immunology ◽

10.3389/fimmu.2020.601895 ◽

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.

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P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings

Journal of Bacteriology ◽

10.1128/jb.185.5.1693-1700.2003 ◽

2003 ◽

Vol 185 (5) ◽

pp. 1693-1700 ◽

Cited By ~ 12

Author(s):

Li Zhao ◽

Shuji Kanamaru ◽

Chatree'chalerm Chaidirek ◽

Fumio Arisaka

Keyword(s):

Electron Microscopy ◽

Gene Product ◽

Analytical Ultracentrifugation ◽

Bacteriophage T4 ◽

Sedimentation Equilibrium ◽

Gene Products ◽

Oligomeric State ◽

Polypeptide Chains ◽

Circular Dichroïsm

ABSTRACT Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.

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Matrix-free hydrodynamic study on the size distribution and conformation of three technical lignins from wood and non-wood

Holzforschung ◽

10.1515/hf-2014-0318 ◽

2016 ◽

Vol 70 (2) ◽

pp. 117-125 ◽

Cited By ~ 3

Author(s):

Qushmua E. Alzahrani ◽

Gary G. Adams ◽

Richard B. Gillis ◽

Tabot M.D. Besong ◽

M. Samil Kök ◽

...

Keyword(s):

Molecular Weight ◽

Analytical Ultracentrifugation ◽

Average Molecular Weight ◽

Sedimentation Equilibrium ◽

Viscosity Data ◽

Oligomeric State ◽

Recent Developments ◽

Plate Shape ◽

High Degree ◽

Technical Lignins

Abstract Molecular weight (MW) and related conformational data of three commercially available technical lignins (Alcell L, kraft L, and soda L) have been studied by means of analytical ultracentrifugation, taking advantage of some recent developments in both sedimentation velocity and sedimentation equilibrium determinations. The lignins were dissolved in dimethyl sulphoxide (with ca. 90% solubility), and solutions were studied with regards to their oligomeric state, heterogeneity profiles (distribution of sedimentation coefficients), and molecular weight distributions (MWD). Alcell L and soda L have similar properties showing one major low MW component and two minor high MW components, whereas kraft L appears to be larger and more uniform, i.e., it shows a more monodisperse MWD. Weight average molecular weight (Mw) data from sedimentation equilibrium obtained by the new SEDFIT-MSTAR procedure in conjunction with MULTISIG analysis were found to be ~18 kDa (Alcell L), 25 kDa (kraft L), and 15 kDa (soda L). Further analysis of the data by means of the routines MULTISIG and M_INVEQ confirmed the presence of additional components in Alcell L and soda L, and the larger size and high degree of monodispersity of kraft L. The intrinsic viscosity data of the three lignins were found to be very similar in the range of 22–24 ml g-1, and all data were consistent with an elongated plate shape molecular structure with an equivalent discoid aspect ratio ~30.

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Terpene polyacrylate TPA5 shows favorable molecular hydrodynamic properties as a potential bioinspired archaeological wood consolidant

Scientific Reports ◽

10.1038/s41598-021-86543-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Michelle Cutajar ◽

Fabrizio Andriulo ◽

Megan R. Thomsett ◽

Jonathan C. Moore ◽

Benoit Couturaud ◽

...

Keyword(s):

Molar Mass ◽

Analytical Ultracentrifugation ◽

Gel Filtration ◽

Axial Ratio ◽

Sedimentation Equilibrium ◽

Archaeological Wood ◽

Molecular Hydrodynamic ◽

Pine Trees ◽

Minimal Concentration ◽

Hydrogen Bonding Potential

AbstractThere is currently a pressing need for the development of novel bioinspired consolidants for waterlogged, archaeological wood. Bioinspired materials possess many advantages, such as biocompatibility and sustainability, which makes them ideal to use in this capacity. Based on this, a polyhydroxylated monomer was synthesised from α-pinene, a sustainable terpene feedstock derived from pine trees, and used to prepare a low molar mass polymer TPA5 through free radical polymerisation. This polymer was extensively characterised by NMR spectroscopy (chemical composition) and molecular hydrodynamics, primarily using analytical ultracentrifugation reinforced by gel filtration chromatography and viscometry, in order to investigate whether it would be suitable for wood consolidation purposes. Sedimentation equilibrium indicated a weight average molar mass Mw of (4.3 ± 0.2) kDa, with minimal concentration dependence. Further analysis with MULTISIG revealed a broad distribution of molar masses and this heterogeneity was further confirmed by sedimentation velocity. Conformation analyses with the Perrin P and viscosity increment ν universal hydrodynamic parameters indicated that the polymer had an elongated shape, with both factors giving consistent results and a consensus axial ratio of ~ 4.5. These collective properties—hydrogen bonding potential enhanced by an elongated shape, together with a small injectable molar mass—suggest this polymer is worthy of further consideration as a potential consolidant.

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