Analysis of heterologous interacting systems by sedimentation velocity: curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and kinetic constants

Stafford, Walter F.; Sherwood, Peter J.

doi:10.1016/j.bpc.2003.10.028

Cited by 243 publications

(269 citation statements)

References 12 publications

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“…To appear as distinct species by sedimentation, the different forms must be meta-stable on the order of at least 10s of minutes (i.e., all equilibria are slow). 56 …”

Section: Discussionmentioning

confidence: 99%

Cutting Edge Structural Protein from the Jaws of Nereis virens

et al. 2008

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The fang-like jaws of the marine polychaete Nereis Virens possess remarkable mechanical properties considering their high protein content and lack of mineralization. Hardness and stiffness properties in the jaw tip are comparable to human dentin and are achieved by extensive coordination of Zn 2+ by a histidine-rich protein framework. In the present study, the predominant protein in the jaw tip, NVjp-1, was purified and characterized by partial peptide mapping and molecular cloning of a partial cDNA from a jaw pulp library. The deduced amino acid sequence revealed an ∼38 kDa histidine-rich protein rich in glycine and histidine (∼36 and 27%, respectively) with no well-defined repetitive motifs. The effects of pH and metal treatment on aggregation, secondary structure, and hydrodynamic properties of recombinant Nvjp-1 are described. Notably, Zn treatment induced the formation of amyloid-like fibers.

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“…To appear as distinct species by sedimentation, the different forms must be meta-stable on the order of at least 10s of minutes (i.e., all equilibria are slow). 56 …”

Section: Discussionmentioning

confidence: 99%

Cutting Edge Structural Protein from the Jaws of Nereis virens

et al. 2008

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“…Concentration was monitored by interference. Data were analyzed globally with the program Sedanal (37). Sednterp was used to calculate partial specific volumes from the amino acid sequences and solvent density from its composition (38).…”

Section: Methodsmentioning

confidence: 99%

Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

et al. 2008

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The spindle pole body (SPB) is a multiprotein complex that organizes microtubules in yeast. Due to its large size and association with the nuclear membrane, little is known about its detailed structure. In particular, although many SPB components and some of the interactions between them have been identified, the molecular details of how most of these interactions occur are not known. The prevalence of predicted coiled-coil regions in SPB proteins suggests that some interactions may occur via coiled coils. Here this hypothesis is supported by biochemical characterization of isolated coiled-coil peptides derived from SPB proteins. Formation of four strongly self-associating coiled-coil complexes from Spc29, Spc42, and Spc72 was demonstrated by circular dichroism (CD) spectroscopy and a fluorescence resonance energy transfer (FRET) assay. Many weaker self-and heteroassociations were also detected by CD, FRET, and/or cross-linking. The thermal stabilities of nine candidate homooligomers were assessed; six unfolded cooperatively with melting temperatures ranging from <11 to >50 °C. Solution studies established that coiled-coil peptides derived from Spc42 and Spc72 form parallel dimers, and this was confirmed for Spc42 by a high-resolution crystal structure. These data contribute to a growing body of knowledge that will ultimately provide a detailed model of the SPB structure.

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“…The optical systems on the analytical ultracentrifuge supply the radial concentration distribution at time intervals during the course of an experiment, c(r,t), and the instrument provides the rotor speed, ω. The quantities sought in a velocity sedimentation experiment are s and D. There are no exact solutions to the Lamm equation: approximate (Behlke and Ristau, 1997;Philo, 1994) and numerical (Demeler and Saber, 1998;Schuck, 1998;Stafford and Sherwood, 2004) solutions form the basis of many sedimentation velocity analysis programs used to extract s and D from AUC data. By taking the ratio s/D, the frictional contribution to these parameters is removed and the result is proportional to the buoyant molar mass, M b , through the Svedberg equation (3) Both the Lamm and Svedberg equations, as presented above, are starting points for the equations that apply to real chemical systems.…”

Section: A Sedimentation Velocitymentioning

confidence: 99%

“…Analysis of reversible interactions by SV is a complex problem Rivas et al, 1999;Schuck, 2003;Stafford, 2000;Stafford and Sherwood, 2004). However, SV may be the only feasible approach for systems that are intrinsically unstable or that do not come to equilibrium in SE experiments.…”

Section: B Data Analysismentioning

confidence: 99%

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Cole

Lary

Moody

et al. 2008

Biophysical Tools for Biologists, Volume One: In Vitro Techniques

361

267

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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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Analysis of heterologous interacting systems by sedimentation velocity: curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and kinetic constants

Cited by 243 publications

References 12 publications

Cutting Edge Structural Protein from the Jaws of Nereis virens

Cutting Edge Structural Protein from the Jaws of Nereis virens

Analysis of Coiled-Coil Interactions between Core Proteins of the Spindle Pole Body

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

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