PubMed · 4595281
Numerous sucrose gradients can be prepared simultaneously by diffusion in horizontal centrifuge tubes.
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A B Stone. 1974. A simplified method for preparing sucrose gradients.. https://doi.org/10.1042/bj1370117
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It was shown previously that complementation could be a powerful mean to probe protein-protein interactions in the normally tetrameric R67 DHFR. Indeed, mixing complementing inactive dimeric mutants produced active heterotetramers. This approach turned a homo-oligomer into a hetero-oligomer and thus allowed the use of combinatorial assays, a subtle analysis of the association forces, and a precise determination of the equilibrium dissociation constants (K(D)) by titrimetry. However, for some of the complementing pairs, the experimental data implied multiple equilibria involving heterodimers, although no monomers could be detected. Thus, the reactions involved had to be identified to elaborate a suitable model to determine the K(D) of those pairs correctly. That model suggested that homodimers associated rapidly before the protomers could be redistributed in a multiple equilibrium system. Kinetic data confirmed that view. The association data at equilibrium were analyzed by multiple curve fitting with all plausible combinations of parameters. This gave a confidence interval for K(D) that is safer than the usual 67% or 90% confidence interval. Finally, the K(D) of one specific reaction, the dissociation of a heterotetramer with the relevant symmetry into two homodimers could be determined with the relevant model for each complementing pair, although multiple equilibria were present. These K(D) can thus be used as a set of references data to test and improve theoretical methods such as association free energy calculations.
Hydrodynamic bead modelling has been widely used in attempts to assess the 3D conformation of proteins in solution. Initially, simple models employing only a small number of beads were used, with a considerable degree of success. Latterly, high-resolution bead models based upon atomic coordinates have been developed, and much more sophisticated questions can in principle be addressed. A detailed analysis is presented of the errors involved in the generation of such models and associated prediction of (translational friction) parameters, and in the practical measurement of these parameters for comparison. It is shown that in most cases, for a particle of only moderate asymmetry, the errors are such that it is not feasible to determine, on an absolute basis, which of a range of candidate conformers is the "correct" one. However, when the properties of the candidate conformers can be compared in relation to those of a "paradigm conformer", whose structure in solution, on the basis of external evidence, can be accepted as correct, then errors cancel and very precise comparisons become possible. The generation of 3D bead models (and hence 3D data files) for a range of candidate conformers is a simple matter, using the existing program MacBEADS, further facilitated by a 3D display module (pro Fit).
Attempts have been made to correlate estimates of molecular weight for a group of cationic polysaccharides known as chitosans between the highly popular technique of size-exclusion chromatography coupled to multi-angle laser light scattering, "SEC-MALLS", and the less convenient but more established technique of sedimentation equilibrium in the analytical ultracentrifuge. Four pharmaceutical grade chitosans of various molecular weights and degrees of acetylation (4-30%) were chosen. Better correlation than previous was achieved, although some batch variability was observed. Despite the broad spectrum in degree of acetylation, a log s degrees(20,w) versus log Mw scaling plot appeared to fit a straight line with power-law exponent b=0.25 +/- 0.04, i.e. between the limits of rod (0.15) and coil (0.4-0.5), although this may be the average of a lower b value at low Mw and higher b at high Mw. With regard to viscosity, a logeta versus logMw scaling plot appeared to also fit a straight line with power-law exponent a=0.96 +/- 0.10, again between the coil (0.5-0.7) and rod (1.8) limits.