A comparison of weight average and direct boundary fitting of sedimentation velocity data for indefinite polymerizing systems

Sontag, Christopher A.; Stafford, Walter F.; Correia, John J.

doi:10.1016/j.bpc.2003.10.029

Cited by 34 publications

(41 citation statements)

References 52 publications

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“…More recently, direct boundary fitting methods for interacting systems have been implemented in SEDANAL, SEDPHAT and ULTRASCAN. When compared, this approach gives comparable results to those obtained using weight-average analysis (Sontag et al, 2004). Some recent examples of direct boundary analysis to define the energetics of associating systems can be found in (Connaghan-Jones et al, 2006;Correia et al, 2005;Gelinas et al, 2004;Snyder et al, 2004).…”

Section: B Data Analysismentioning

confidence: 66%

“…An advantage of this method is that the weight average sedimentation coefficient is a thermodynamically valid parameter that is determined the sample composition in the plateau and is independent of the kinetics of the interactions, provided that the sample is at equilibrium prior to sedimentation. Examples of this approach can be found in studies of CMV protease dimerization (Cole, 1996) and the complex association reactions of tubulin (Correia, 2000;Sontag et al, 2004) and HIV rev (Surendran et al, 2004). Interacting systems can also be characterized calculation of g(s*) distributions using the time-derivative method (Stafford, 2000).…”

Section: B Data Analysismentioning

confidence: 99%

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Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Cole

Lary

Moody

et al. 2008

Methods in Cell Biology

368

274

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Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution. AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations. Three optical systems are available for the analytical ultracentrifuge (absorbance, interference and fluorescence) that permit precise and selective observation of sedimentation in real time. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules in complex mixtures and at high solute concentrations. In sedimentation velocity, the movement of solutes in high centrifugal fields is interpreted using hydrodynamic theory to define the size, shape and interactions of macromolecules. Sedimentation equilibrium is a thermodynamic method where equilibrium concentration gradients at lower centrifugal fields are analyzed to define molecule mass, assembly stoichiometry, association constants and solution nonideality. Using specialized sample cells and modern analysis software, researchers can use sedimentation velocity to determine the homogeneity of a sample and define whether it undergoes concentration-dependent association reactions. Subsequently, more thorough model-dependent analysis of velocity and equilibrium experiments can provide a detailed picture of the nature of the species present in solution and their interactions.

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Section: B Data Analysismentioning

confidence: 66%

Section: B Data Analysismentioning

confidence: 99%

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Cole

Lary

Moody

et al. 2008

Methods in Cell Biology

368

274

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show abstract

“…Tubulin undergoes Mg 2þ -and nucleotide-dependent oligomerization under conditions that do not favor microtubule assembly (27). This serves as a baseline reaction for proper interpretation of all other association reactions that might occur in a drug-dependent manner.…”

Section: Resultsmentioning

confidence: 99%

“…All experiments were initially analyzed with DCDT þ2 to produce g(s) distributions and weight-average S values (Ŝ w ) (29). Quantitative analysis ofŜ w data (26) or boundary shape (31) involves fitting to a model for indefinite polymerization (either tubulin alone or ligand-mediated polymer growth) where K iso is the propagation constant for polymer growth in the presence or absence of drug (V for vinca alkaloid; see refs (26)(27)(28) for details).…”

mentioning

confidence: 99%

Macromolecular Interaction of Halichondrin B Analogues Eribulin (E7389) and ER-076349 with Tubulin by Analytical Ultracentrifugation

Alday

Correia

2009

Biochemistry

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Halichondrin B is an antimitotic drug that inhibits microtubule assembly. To understand the molecular details of its interaction with tubulin, we investigated the binding of two halichondrin B analogues, eribulin (previously, ER-086526, E7389) and ER-076349, to tubulin by quantitative analytical ultracentrifugation. Eribulin is currently undergoing phase III clinical trials for cancer; ER-076349 is a closely related analogue with C.35 hydroxyl instead of C.35 primary amine [Towle, M. J., et al. (2001) Cancer Res. 61, 1013]. Below the critical concentration for microtubule assembly and in the presence of GDP, tubulin undergoes weak self-association into short curved oligomers. Eribulin inhibits this oligomer formation 4-6-fold, while ER-076349 slightly stimulates oligomer formation by 2-fold. This is in contrast to vinblastine which strongly stimulates large spiral polymers by 1000-fold under these same conditions. Vinblastine-induced spiral formation is strongly inhibited by both eribulin and ER-076349. Colchicine binding to the intradimer interface has no significant effect on small oligomer formation or the inhibitory activity of eribulin on this process. These results suggest that halichondrin B analogues bind to the interdimer interface or to the beta-subunit alone, disrupt polymer stability, and compete with vinblastine-induced spiral formation. Stathmin is known to form a tight 1:2 complex with tubulin. Eribulin strongly inhibits formation of the 1:2 stathmin-tubulin complex (>3.3 kcal/mol), while ER-076349 weakens formation of the 1:2 complex by approximately 1.9 kcal/mol. These results suggest that eribulin is a global inhibitor of tubulin polymer formation, disrupting tubulin-tubulin contacts at the interdimer interface. ER-076349 also perturbs tubulin-tubulin contacts, but in a more polymer specific manner, reflecting adaptability of the interdimer interface to drug and polymer polymorphism. These results suggest halichondrin B analogues exhibit unique tubulin-based activities that may underlie the clinical utility of these compounds.

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“…From Table 1 it is apparent that the sedimentation velocity data for SEPT11C is more compatible with a monomer at lower concentrations and a dimer at higher concentrations. In our analysis we therefore assumed a dynamic equilibrium between these two states in solution with all changes in s 20,w being due to the monomer/dimer ratio (24)(25)(26)(27). The K Dapp of 7.2 5 0.8 mM for the SEPT11C homodimer was determined by fitting the data to the Boltzmann equation (Fig.…”

Section: Analytical Ultracentrifugationmentioning

confidence: 99%

Heterotypic Coiled-Coil Formation is Essential for the Correct Assembly of the Septin Heterofilament

Sala

Valadares

Macêdo

et al. 2016

Biophysical Journal

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Protein-protein interactions play a critical role in promoting the stability of protein quaternary structure and in the assembly of large macromolecular complexes. What drives the stabilization of such assemblies is a central question in biology. A limiting factor in fully understanding such systems is the transient nature of many complexes, making structural studies difficult. Septins comprise a conserved family of guanine nucleotide binding proteins that polymerize in the form of heterofilaments. In structural terms, they have a common organization: a central GTPase domain, an N-terminal domain, and a C-terminal domain; the latter is predicted to form a coiled coil. Currently, even for the best characterized human septin heterocomplex (SEPT2/SEPT6/SEPT7), the role of C-terminal domain is not fully established, and this is partly due to the absence of electron density for the C-terminal domains in the x-ray structure. Here we present results on the homo/heterotypical affinity for the C-terminal domains of human septins belonging to the SEPT6 and SEPT7 groups (SEPT6C/8C/10C/11C and SEPT7C, respectively) and provide clear evidence that this domain determines the preference for heterotypic interactions at one specific interface during the assembly of the heterofilament. This observation has wider implications where macromolecular assemblies are defined by coiled-coil protein interactions.

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A comparison of weight average and direct boundary fitting of sedimentation velocity data for indefinite polymerizing systems

Cited by 34 publications

References 52 publications

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Macromolecular Interaction of Halichondrin B Analogues Eribulin (E7389) and ER-076349 with Tubulin by Analytical Ultracentrifugation

Heterotypic Coiled-Coil Formation is Essential for the Correct Assembly of the Septin Heterofilament

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