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  • 1
    ISSN: 1432-1017
    Keywords: NMR ; Dynamics ; Src-homology domain ; Secondary structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of1H-15N-correlated 2D HSQC,15N-edited 3D TOCSY-HMQC, and15N-edited 3D NOESY-HMQC spectra. By analysis of the α-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain. To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.
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  • 2
    ISSN: 1432-1017
    Keywords: GAL4 fragments ; Apparent dissociation constants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract It has previously been suggested that the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 exists in solution in dimeric form, with the region responsible for dimerisation somewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 followed by subsequent characterization by equilibrium sedimentation in the analytical ultracentrifuge have been used to define more precisely the regions required for DNA binding and protein self-association. Sedimentation equilibrium analyses confirmed that the ‘hydrophobic region’ of the protein (residues 54–97, which contains a larger proportion of α-helix), is essential for dimerisation, with an apparent dissociation constant KD,app, of ≈50 μM for the 1–94 residue peptide and ≈20 μM for the 1–147 residue peptide. Our studies do not rule out the possible formation of small amounts of additional higher order complexes.
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  • 3
    ISSN: 1432-1017
    Keywords: Trimethylamine dehydrogenase ; Analytical ultracentrifugation ; Hydrodynamics ; Homodimers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Earlier studies using x-ray crystallography have shown that trimethylamine dehydrogenase (TMADH) from methylotropic bacteria exists as homodimers in the crystalline state. In this present hydrodynamic study we show that this is true also in dilute solution conditions and investigate the degree of swelling or relaxation of the protein in solution. Analytical ultracentrifugation was used to determine the molar mass and to investigate whether the homodimeric nature of this molecule in crystal form — as visualized by x-ray crystallography — is reproduced in dilute solution at temperatures between 4 and 40°C. The globular solution structure determined at 4 and 40°C is in good agreement with crystallographic data although trimethylamine dehydrogenase was found to be either more asymmetric in solution — or highly hydrated —, a phenomenon found to increase with temperature. In agreement with the crystallographic structure, the enzyme sediments as a homodimer with a molar mass of (163,000±5,000) g/mol. The concentration dependence of the sedimentation coefficient in the range of 0–1 mg/ml, indicates that no association or dissociation occurs. These findings are additionally supported by sedimentation equilibrium data in the concentration range of 0 to 1.8 mg/ml. Finally, from the sedimentation coefficient distribution at various temperatures, it was concluded that the enzyme is conformationally flexible and assumes an even more expanded structure at higher temperatures which is in good agreement with the hydrodynamic calculations performed.
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  • 4
    ISSN: 1432-1017
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
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  • 5
    ISSN: 1432-1017
    Keywords: Key words Glutamate dehydrogenase ; Analytical ultracentrifugation ; Allostery ; Quaternary structure ; Subunit communication
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an s20,w value for D165H of (6.07 ± 0.01)S which compares with (11.08 ± 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.
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  • 6
    ISSN: 1432-1017
    Keywords: Key words T-lymphocytes ; Dissociation constant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract CD2 is a cell adhesion molecule found on the plasma membrane of T-lymphocytes. Its counter-receptor in rat is the structurally related CD48. This interaction is believed to contribute to the adhesion of T-cells to other cells such as cytotoxic targets and antigen presenting cells. Cell-cell adhesion involves the formation of multiple cell adhesion molecule complexes at the cell surface and if cell-cell de-adhesion is to occur, these complexes need to be disrupted. The affinities of cell adhesion molecule interactions are suggested to be relatively weak to allow this de-adhesion of cell-cell interactions. The CD2/CD48 interaction has been studied using recombinant extracellular proteins and the affinity of the interaction of soluble recombinant rat CD2–CD48 has been determined (at 37°C) using surface plasmon resonance (and shown to be weak), with the dissociation constant Kd=60–90 µm. The values determined by surface plasmon resonance results could be affected by the immobilisation of the ligand on the chip and any self-association on the chip. We used three different analytical ultracentrifuge procedures which each allowed the interaction to be studied in free solution without the need for an immobilisation medium. Both sedimentation equilibrium (using direct analysis of the concentration distribution and also modelling of molecular weight versus concentration data) and sedimentation velocity at 5°C yielded dissociation constants in the range of 20– 110 µm, supporting the surface plasmon resonance findings showing that binding between these cell adhesion molecules is relatively weak. These studies also ruled out the presence of any significant self-association of the reactants which could lead to systematic error in the surface plasmon resonance results.
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  • 7
    ISSN: 1432-1017
    Keywords: Key words GroEL ; Chaperonin ; Ultracentrifugation ; Sedimentation equilibrium ; Sedimentation velocity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract A combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge is used to investigate the hydrodynamic integrity and increased self-association interactions of the mutant GroEL Y203W when compared to the wild-type GroEL molecule, which may be derived from increased hydrophobic exposure caused by the mutation. Sedimentation velocity has revealed that three distinct species were present throughout the concentration ranges used, corresponding to 14-mer (GroEL “super monomer”) and 28-mer (“super dimer”) subunit compositions with a small amount of 42-mer (“super trimer”), which, from the relative concentration of each species, would give an estimated weight average molecular weight of (1.0 ± 0.1) × 106 Da. Sedimentation equilibrium gave an apparent weight average molecular weight (M w,app) of (910,000 ± 5000) Da, which is in agreement with these findings. These results are in contrast to wild-type GroEL which, in excellent agreement with the previous findings of Behlke and co-workers, revealed a single species with an M w,app of (805,000 ± 5200) Da and a sedimentation coefficient s 0 20,w of (21.6 ± 0.3) S. We therefore conclude that the tryptophan mutation at the Y203 location causes a significant degree of self-association of the GroEL 14-mer assembly (with dimer and trimer present). These findings would appear to correlate well with the findings of Gibbons et al., who showed an increase in hydrophobic exposure due to this mutation.
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  • 8
    ISSN: 1432-1017
    Keywords: Key words Hydrodynamics programs ; Bead modelling ; Fluorescence ; Birefringence ; Scattering
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Single-valued hydrodynamic coefficients of a rigid particle can be calculated from existing theories and computer programs for either bead models or ellipsoids. Starting from these coefficients, we review the procedures for the calculation of complex solution properties depending on rotational diffusion, such as the decays of electric birefringence and fluorescence anisotropy. We also describe the calculation of the scattering form factor of bead models. The hydrodynamic coefficients and solution properties can be combined to give universal, shape-dependent functions, which were initially intended for ellipsoidal particles, and are extended here for the most general case. We have implemented all these developments in a new computer program, SOLPRO, for calculation of SOLution PROperties, which can be linked to existing software for bead models or ellipsoids.
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  • 9
    ISSN: 1432-1017
    Keywords: Key words Band 3 ; Membrane protein ; Analytical ultracentrifugation ; Hydrodynamics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract The dilute solution behaviour of the transmembrane domain (TMD) of the human erythrocyte anion exchanger Band 3 was studied by analytical ultracentrifugation. Sedimentation velocity and equilibrium studies of the TMD solubilized with the detergent C12E8 demonstrate that the protein is a stable dimer in the concentration range 0.1 to 1 mg/ml. There is no evidence of a dissociation at low concentrations or of an association at higher concentrations. Hydrodynamic calculations applying a prolate ellipsoid of revolution and assuming a hydration of w=0.35 result in an asymmetrical particle with an axial ratio (a/b) of ∼3.5.
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  • 10
    ISSN: 1432-1017
    Keywords: Key words Analytical ultracentrifugation ; Sedimentation equilibrium ; MSTAR function ; Molar mass determination ; Polydispersity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract This paper describes a program available for PC's for the evaluation of molecular weights from sedimentation equilibrium. This program, in its two forms – MSTARA for absorption optical records and MSTARI for interference optical records – requires no prior assumption of the nature of the system (ideal, non-ideal, monodisperse, polydisperse, self-associating etc.) and takes into consideration the whole solute distribution (i.e. from solution meniscus to cell base) in the ultracentrifuge cell rather than just a selected data-set. MSTARA or MSTARI are therefore recommended as a first analysis programme of sedimentation equilibrium data coming off an absorption or interference based analytical ultracentrifuge. These programmes are therefore particularly well suited if heterogeneity (polydispersity or interaction phenomena) or non-ideality is suspected. Their use is demonstrated for a series of data-set types (ideal, non-ideal, polydisperse and self-associating). Although MSTARA and MSTARI are model independent, they provide the basis for more detailed analysis of interactions, polydisperse distributions or non-ideality via easy export of ASCII datafiles to model dependent routines.
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