Ultracentrifuge studies with absorption optics

Schachman, H. K.; Gropper, L.; Hanlon, Sue; Putney, F.

doi:10.1016/0003-9861(62)90259-x

Cited by 99 publications

(27 citation statements)

References 11 publications

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“…Absorption optics allow us to selectively monitor labeled profilin in the presence of other proteins, to determine directly the distributions of free and bound profilin, and to calculate equilibrium binding constants (Schachman, 1960;Schachman et al, 1962). A similar method has been used previously to measure equilibrium binding of pyrene-labeled actobindin to actin (Bubb et al, 1994 where P o and P 1 are the fitting constants to be determined, r is radial position, r m is the radius of the meniscus, and p and pa are the effective reduced molecular weights of profilin alone and profilin bound to Arp2/3 complex or to actin monomers.…”

Section: Profilin Binding By Equilibrium Ultracentrifugationmentioning

confidence: 99%

Arp2/3 Complex fromAcanthamoebaBinds Profilin and Cross-links Actin Filaments

Mullins

Kelleher

et al. 1998

MBoC

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The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motileAcanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

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Section: Profilin Binding By Equilibrium Ultracentrifugationmentioning

confidence: 99%

Arp2/3 Complex fromAcanthamoebaBinds Profilin and Cross-links Actin Filaments

Mullins

Kelleher

et al. 1998

MBoC

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“…The pioneering work of Schachman and colleagues (19,20) in the development of an absorbance optical system for the analytical ultracentrifuge made possible the selective detection of macromolecular components, which has proven to be extremely useful in the study of proteins and protein interactions. In 1966, Steinberg and Schachman (21) developed the simultaneous detection of absorbance and refractive index profiles for the analysis of protein-small molecule interactions in terms of constituent effective molecular weights and constituent sedimentation coefficients.…”

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confidence: 99%

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation

Balbo

Minor

Velikovsky

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

125

154

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Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.protein interactions ͉ size distribution

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“…Since 1960 the ultracentrifuge has been used increasingly for the study ofinteractions between large and small molecules, particularly by Schachman and co-workers (Schachman, Gropper, Hanlon & Putney, 1962;Schachman, 1963;Steinberg & Schachman, 1966). In these studies photoelectric scanning devices have been used for following the changes in light-absorption that occur when the interacting system of macromolecule and ligand is subjected to a high centrifugal field.…”

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confidence: 99%

Sedimentation studies on the interaction of proflavine with deoxyribonucleic acid

Lloyd

Prutton

Peacocke

1968

Biochemical Journal

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A method is described of using photography to measure the concentrations of a small ligand (proflavine) above and below the boundary of a macromolecule (DNA, both native and denatured) sedimenting in the ultracentrifuge. The measurements are used to determine the extent of the binding of proflavine to DNA, and the results compared with those obtained by a spectrophotometric method. The results obtained by the two methods agree within 10%, thus validating the spectrophotometric technique under these conditions. The variation of the sedimentation coefficient with the extent of binding of proflavine was also studied. The results are discussed in relation to previously observed changes in the viscosity of the solutions.

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Ultracentrifuge studies with absorption optics

Cited by 99 publications

References 11 publications

Arp2/3 Complex fromAcanthamoebaBinds Profilin and Cross-links Actin Filaments

Arp2/3 Complex fromAcanthamoebaBinds Profilin and Cross-links Actin Filaments

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation

Sedimentation studies on the interaction of proflavine with deoxyribonucleic acid

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