VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.

VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N- and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures.

Colloidal properties of human transferrin receptor in detergent free solution.

The colloidal properties of transferrin receptor, isolated from human placenta, in detergent free solution has been investigated by light scattering techniques and analytical ultracentrifugation. In detergent free solution at 293.2 K, hTfR forms stable aggregates with an apparent hydrodynamic radius of 17 nm. The molecular mass was determined by ultracentrifugation to lie between (1722+/-87) kDa (sedimentation equilibrium) and (1675+/-46) kDa (sedimentation velocity). This implies that the aggregates are build up from nine hTfR dimers. Based on model calculations, which are in good agreement with the experimental data, we propose a torus-like structure for the aggregates. Upon pH shift from pH 7.5 to 5.0 or removal of the N-linked carbohydrate chains, formation of larger aggregates is induced. These aggregates can be described in terms of porous fractal structures. We propose a simple model, which accounts for that behaviour assuming that the aggregation is mainly due to the reduction of negative surface charge.

Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer.

The CopR protein is one of the two regulators of pIP501 copy number. It acts as transcriptional repressor at the essential repR promoter pII. Previously, we found that CopR contacts two consecutive major grooves (site I and site II) on the same face of the DNA. In spite of identical sequence motifs in these sites, neighboring bases were contacted differently. Furthermore, we showed that CopR can dimerize in solution. We demonstrate by two independent methods that CopR binds the DNA as a dimer. We present data that suggest that the sigmoidal CopR-DNA binding curve published previously is the result of two coupled equilibria: dimerization of CopR monomers and CopR dimer-DNA binding. A KD-value of 1.44(+/-0.49)x10(-6) M for CopR dimers was determined by analytical ultracentrifugation. Based on this value and the binding curve, the equilibrium dissociation constant K2 for the CopR-DNA complex was calculated to be 4(+/-1. 3)x10(-10) M. Quantitative Western blot analysis was used to determine the intracellular concentration of CopR in Bacillus subtilis. This value, 20x10(-6) to 30x10(-6) M, is 10 to 20-fold higher than the equilibrium constant for dimer dissociation, suggesting that CopR binds in vivo as a preformed dimer.

Crystallization and X-ray examination of bovine adrenodoxin.

Crystals of adrenodoxin from bovine adrenocortical mitochondria were obtained by the hanging-drop vapor diffusion technique. The crystals belong to a hexagonal crystal lattice with cell parameters 172.50 A and 183.49 A. There are 12 molecules in the asymmetric unit. The crystals diffract to beyond 4.0 A resolution.

Supramolecular structure of the recombinant murine small heat shock protein hsp25.

The size and shape of the recombinant murine small heat shock protein, hsp25, have been analyzed by hydrodynamic and electron microscopic methods. According to these studies recombinant hsp25 exists in large complexes with a sphere-like shape and diameters of 15-18 nm. The molecular mass of these complexes amounts to about 730 kDa indicating that they are composed of about 32 monomers.

Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation.

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5+/-2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)(6). At pH 5.8, (RepA)(6) associates to form a dimer with a molecular mass of 366.2+/-4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)(6) at pH 5.8 and 7.6. The dissociation constants, K(d), for the equilibrium binding of (dA)(30) to the (RepA)(6) dimer at pH 5.8 and to (RepA)(6) at pH 7.6 were determined at 10 degrees C in the presence of 0.5 mM ATPgammaS, 10 mM MgCl(2) and 60 mM NaCl as K(d5.8)=0.94+/-0.13 microM at pH 5.8 and K(d7. 6)=25.4+/-6.4 microM at pH 7.6. The stoichiometries, n, for the two complexes (dA)(30)/(RepA)(6) dimer and (dA)(30)/(RepA)(6) at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)(30) molecule was bound per (RepA)(6) dimer, while at pH 7.6 one (dA)(30) molecule was bound to one (RepA)(6). Binding curves were compatible with a single ssDNA binding site present on the (RepA)(6) dimer and on (RepA)(6), respectively, with no indication of cooperativity. (RepA)(6) tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)(6) dimer was observed both in the absence and presence of (dA)(30).

A 6 bp Z-DNA hairpin binds two Z alpha domains from the human RNA editing enzyme ADAR1.

The Z alpha domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Z alpha domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Z alpha and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (> or = 6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The omega-H(6) protein has a modified conformation with lower alpha-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.

Interaction of fMet-tRNA(fMet) with the C-terminal domain of translational initiation factor IF2 from Bacillus stearothermophilus.

Analytical ultracentrifugation studies indicated that the C-terminal domains of IF2 comprising amino acid residues 520-741 (IF2 C) and 632-741 (IF2 C-2) bind fMet-tRNA with similar affinities (K(d) at 25 degrees C equal to 0.27 and 0.23 microM, respectively). Complex formation between fMet-tRNA(fMet) and IF2 C or IF2 C-2 is accompanied by barely detectable spectral changes as demonstrated by a comparison of the Raman spectra of the complexes with the calculated sum of the spectra of the individual components. These results and the temperature dependence of the K(d) of the protein-RNA complexes indicate that complex formation is not accompanied by obvious conformational changes of the components, and possibly depends on a rather small binding site comprising only a few interacting residues of both components.

Analysis of the thermodynamic non-ideality of proteins by sedimentation equilibrium experiments.

This paper presents a modified method to determine experimentally the second virial coefficient of protein solutions by sedimentation equilibrium experiments. The improvement is based on the possibility of fitting simultaneously up to seven radial concentration distribution curves of solutions with different loading concentrations. The possibility of precise determination of the second virial coefficient allows estimation of the net charge and the excluded volume of a monomeric protein. Application of the method is demonstrated for lysozyme and ovalbumin. In 0.1 M sodium acetate buffer, pH 4.5, the second virial coefficient of hen egg white lysozyme amounts to 24 +/- 1 ml/g. Analysis based on spherical particle theory yield an excluded volume of 3.5 ml/g and a charge dependent value of 20.5 ml/g which is induced by a net charge number of 14.1 +/- 1. Under low salt conditions self-association processes on lysozyme are unfavorable due to electrostatic repulsion. To overcome these repulsive contributions, either a shift to neutral pH or addition of at least 2% NaCl is necessary. In this way the charge dependent contribution decreases below the value responsible for the excluded volume and allows crystallization of the protein. Similar effects can be observed with ovalbumin. The high virial coefficient observed at pH 8.5 is induced by the high net charge number of 27 +/- 1.

An improved approximate solution of the Lamm equation for the simultaneous estimation of sedimentation and diffusion coefficients from sedimentation velocity experiments.

Sedimentation and diffusion coefficients are important parameters to describe size and shape of macromolecules in solution. The data can be obtained from sedimentation velocity experiments by a nonlinear fitting procedure using approximate solutions for the Lamm equation. Here, we present a modification of such a model function that was originally proposed by Fujita [H. Fujita, Mathematical Theory of Sedimentation Analysis, Wiley, New York, 1962]. The extended model function is well suitable to study low molecular mass compounds. The improvement of this solution given here is based on using an adjustable value for the explicit integration variable, z, the reduced radius. This modification leads to more accurate sedimentation and diffusion coefficients compared to using a constant value of 0.5 as used by Fujita. The advantage of our modification was demonstrated by the analysis of noise-free curves calculated using the finite element method, as well as experimental curves obtained for the peptides angiotensin I and II. The relatively low sedimentation and diffusion coefficients found for both substances indicate that the peptides exist as extended chains of about 3.65 nm (angiotensin I) or 3.04 nm length (angiotensin II) in solution. The lack of higher-order structure of the peptides that was derived also from CD spectra might facilitate receptor binding, and could be one reason for the fast proteolytic digestion of the free peptides.

Analysis of protein self-association under conditions of the thermodynamic non-ideality.

Analysis of protein-protein interactions in highly concentrated solutions requires a consideration of the non-ideality in such solutions which is expressed by the virial coefficients. Different equations are presented to estimate effects of the thermodynamic non-ideality on the macromolecular interaction of self-associating proteins in sedimentation equilibrium experiments. Usually the influence of thermodynamic non-ideal behavior are described by concentration power series. The convergence of such power series is limited at high solute concentration. When expressing the thermodynamic non-ideality by an activity power series this disadvantage can be minimized. The developed centrifuge equations are the basis for a global analysis to estimate equilibrium constants and the corresponding thermodynamic activities of the reactants. Based on fit analysis of synthetic concentration profiles it was established that marked deviations from the expected association constants are observed for proteins with strong association forces between solute molecules. Considerable differences were also observed in weakly interacting systems. This was due to the excluded volume of the protein which is similar in magnitude to the binding constant. For interactions with moderate affinities values extremely close to the true binding values were obtained, as confirmed by experimental results with concanavalin A.

Analysis of interacting biopolymer systems by analytical ultracentrifugation.

Many of the functions of biological macromolecules are based on specific interactions. Extended concentration dependent studies of sedimentation coefficients or molecular masses of biopolymers are highly useful for describing the different kinds of association phenomena. These studies allow one to determine the partial concentrations of monomers and associates or reactants and complexes in self-associating systems or heterologous associations, respectively. Furthermore, in combination with corresponding measurements of biological activity these data allow one to estimate the individual activity parameters of components involved in equilibrium processes. The study of self-association and heterologous association using analytical ultracentrifugation, some recent developments therein, and its application to different examples are outlined here.

Dithionite reduced carbon monoxide complex of cytochrome P450cam is a monomer.

Sedimentation experiments on cytochrome P450cam (CYP101) has been performed to compare the molecular mass of the protein in the oxidized state and as carbon monoxide complex. The oxidized protein in the absence of beta-mercaptoethanol is a dimer with a molecular mass of 92 kDa. Addition of mercaptoethanol avoids completely the dimerization. Dithionite reduced P450cam in the presence of carbon monoxide has been found to be a monomeric protein.

Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles.

A new method for the direct molecular mass determination from sedimentation velocity experiments is presented. It is based on a nonlinear least squares fitting procedure of the concentration profiles and simultaneous estimation of the sedimentation and diffusion coefficients using approximate solutions of the Lamm equation. A computer program, LAMM, was written by using five different model functions derived by Fujita (1962, 1975) to describe the sedimentation of macromolecules during centrifugation. To compare the usefulness of these equations for the analysis of hydrodynamic results, the approach was tested on data sets of Claverie simulations as well as experimental curves of some proteins. A modification for one of the model functions is suggested, leading to more reliable sedimentation and diffusion coefficients estimated by the fitting procedure. The method seems useful for the rapid molecular mass determination of proteins larger than 10 kDa. One of the equations of the Archibald type is also suitable for compounds of low molecular mass, probably less than 10 kDa, because this model function requires neither the plateau region nor a meniscus free of solute.