Interaction of the trp RNA-binding attenuation protein (TRAP) with anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5'-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA(Trp) induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP-AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of approximately 3 x 10(14)M(-3). About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP-AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.     

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuumNot only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.     

Effect of Calcium Ions on Hydrodynamic Properties of Pentameric and Decameric C-Reactive Protein in Solution

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s ⁰ _20,w of pentameric C-reactive protein in solution containing 2 mM Ca²⁺ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses M _w and M _z obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca²⁺ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.     

Determination of small differences in buoyant density of macromolecules by analytical density gradient equilibrium centrifugation.

A sensitive method is proposed for the determination of small differences between the buoyant densities of different species of monodisperse macromolecules by analytical density gradient equilibrium centrifugation. The procedure involves the measurement at sedimentation equilibrium of the bandwidths of the concentration distribution of the separate macromolecules and of a mixture of the different species. The difference in buoyant densities can then be estimated from the difference between the bandwidths.     

Mechanism of PKR activation: dimerization and kinase activation in the absence of double-stranded RNA

The kinase PKR is a central component of the interferon antiviral pathway. PKR is activated upon binding double-stranded (ds) RNA to undergo autophosphorylation. Although PKR is known to dimerize, the relationship between dimerization and activation remains unclear. Here, we directly characterize dimerization of PKR in free solution using analytical ultracentrifugation and correlate self-association with autophosphorylation activity. Latent, unphosphorylated PKR exists predominantly as a monomer at protein concentrations below 2 mg/ml. A monomer sedimentation coefficient of s(20,w)(0)=3.58 S and a frictional ratio of f/f(0)=1.62 indicate an asymmetric shape. Sedimentation equilibrium measurements indicate that PKR undergoes a weak, reversible monomer-dimer equilibrium with K(d)=450 microM. This dimerization reaction serves to initiate a previously unrecognized dsRNA-independent autophosphorylation reaction. The resulting activated enzyme is phosphorylated on the two critical threonine residues present in the activation loop and is competent to phosphorylate the physiological substrate, eIF2alpha. Dimer stability is enhanced by approximately 500-fold upon autophosphorylation. We propose a chain reaction model for PKR dsRNA-independent activation where dimerization of latent enzyme followed by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by activated enzyme within high-affinity heterodimers. Our results support a model whereby dsRNA functions by bringing PKR monomers into close proximity in a manner that is analogous to the dimerization of free PKR.     

A revised equation relating DNA buoyant density to guanine plus cytosine content

An equation relating DNA buoyant density of CsCl to G + C content is given which uses the correct density value of Escherichia coli DNA as the reference. This is done to eliminate the current confusion brought about by two references states.     

Modulation of quaternary structure of S100 proteins by calcium ions

It is well established that calcium binding leads to conformational changes in S100 proteins. These conformational changes are thought to activate the protein and render a protein conformation that is capable of binding other proteins. The basic quaternary structural motif of S100 proteins is a homodimer, however there is little information if higher order non-covalent oligomers are also formed and whether these oligomers are of functional relevance. To this end we performed equilibrium analytical ultracentrifugation experiments for 16 S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7, S100A8, S100A9, S100A10, S100A11, S100A12, S100A13, S100B, S100P, and S100Z) under reducing conditions in the absence and presence of calcium ions. We show that the addition of calcium promotes the formation of tetrameric structures which could be further enhanced under in vivo conditions where there is an additional effect of molecular crowding.     

Solution structure of the complex between CR2 SCR 1-2 and C3d of human complement: an X-ray scattering and sedimentation modelling study

Complement receptor type 2 (CR2, CD21) forms a tight complex with C3d, a fragment of C3, the major complement component. Previous crystal structures of the C3d-CR2 SCR 1-2 complex and free CR2 SCR 1-2 showed that the two SCR domains of CR2 form contact with each other in a closed V-shaped structure. SCR 1 and SCR 2 are connected by an unusually long eight-residue linker peptide. Medium-resolution solution structures for CR2 SCR 1-2, C3d, and their complex were determined by X-ray scattering and analytical ultracentrifugation. CR2 SCR 1-2 is monomeric. For CR2 SCR 1-2, its radius of gyration R(G) of 2.12(+/-0.05) nm, its maximum length of 10nm and its sedimentation coefficient s20,w(o) of 1.40(+/-0.03) S do not agree with those calculated from the crystal structures, and instead suggest an open structure. Computer modelling of the CR2 SCR1-2 solution structure was based on the structural randomisation of the eight-residue linker peptide joining SCR 1 and SCR 2 to give 9950 trial models. Comparisons with the X-ray scattering curve indicated that the most favoured arrangements for the two SCR domains corresponded to an open V-shaped structure with no contacts between the SCR domains. For C3d, X-ray scattering and sedimentation velocity experiments showed that it exists as a monomer-dimer equilibrium with a dissociation constant of 40 microM. The X-ray scattering curve for monomeric C3d gave an R(G) value of 1.95 nm, and this together with its s20,w(o) value of 3.17 S gave good agreement with the monomeric C3d crystal structure. Modelling of the C3d dimer gave good agreements with its scattering and ultracentrifugation parameters. For the complex, scattering and ultracentrifugation experiments showed that there was no dimerisation, indicating that the C3d dimerisation site was located close to the CR2 SCR 1-2 binding site. The R(G) value of 2.44(+/-0.1) nm, its length of 9 nm and its s20,w(o) value of 3.45(+/-0.01) S showed that its structure was not much more elongated than that of C3d. Calculations with 9950 models of CR2 SCR 1-2 bound to C3d through SCR 2 showed that SCR 1 formed an open V-shaped structure with SCR 2 and was capable of interacting with the surface of C3d. We conclude that the open V-shaped structures formed by CR2 SCR 1-2, both when free and when bound to C3d, are optimal for the formation of a tight two-domain interaction with its ligand C3d.