Ann Ginsburg

Molecular-weight determinations during the approach to sedimentation equilibrium.

Abstract Discussed herein are different methods for treating the data obtained in an ultracentrifugal study during the approach to sedimentation equilibrium. All of these methods are based on the original proposal of Archibald. Equations are presented and the advantages and disadvantages of the various procedures are illustrated with results from experiments on purified proteins and known mixtures of homogeneous materials. Since it is difficult to obtain reliable data at the bottom of existing ultracentrifuge cells, special efforts have been directed toward this problem. It was found that the addition of a small amount of a dense, inert, transparent liquid, such as silicone fluid, to the ultracentrifuge cell facilitates the determination of precise molecular weights at the bottom of the cell. In effect, this dense liquid creates in the ultracentrifuge cell a false bottom of the correct shape. Moreover, the transparency of this cell bottom greatly simplifies the detection of aggregated material in the solution. Precise values of the molecular weights of ribonuclease and β-lactoglobulin have been obtained, and the results are in excellent agreement with values derived by other methods. Furthermore, correct weight-average molecular weights have been determined for various known mixtures of these proteins. It was also found that inhomogeneity can be readily demonstrated. For certain types of mixtures the Archibald method gives not only accurate values of the weight-average molecular weight but it also provides a reasonable estimate of the molecular weight of the heavy component.

Carmil is a bonafide capping protein interactant

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the α- and β-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ∼0.4 μm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ∼200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer ↔ dimer equilibrium with an association constant of 1.0 × 106 m-1. Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (∼1 and 2 μm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.

Temperature and guanidine induced unfolding of dodecameric glutamine synthetase from E. coli

Glutamine synthetase (GS) of 622000 Mr from E. coli is composed of 12 identical subunits which are structurally arranged in two superimposed hexagonal rings with active sites at subunit interfaces. The enzyme undergoes reversible, thermally induced, partial unfolding without dissociation of subunits at pH 7 in the presence of 100 mM KC1 and 1.0 mM MnC12. Cooperative interactions link partial unfolding reactions of all subunits within the Mn*GS dodecamer (AHcal = 750 kJ/mol) and only &Q, two-state transitions with similar Tm values (324+2 K) are observed. Enthalpies at 310 K for subunit dissociation and subsequent unfolding were estimated to be -61 and -55 J/g, respectively, or -100-fold the value of AH for thermal unfolding. Differential scanning calorimetry (DSC) and temperature-induced spectral changes of oligomeric proteins give information on the cooperativity of thermally induced unfolding reactions. To apply reversible thermodynamic treatments of DSC data obtained with oligomeric proteins, we should know the following: (1) Unfolding transitions are reversible and the measured parameters (Tm and AHcal) are independent of the scan rate used to collect the data. (2) What is the molecular species undergoing unfolding (i.e., can the moles of biopolymer be defined)? (3) Can pre- and post-transitional baselines be precisely drawn and extrapolated to a reasonable ACp value? (4) How many thermodynamic domains (AHcal/AHv~) or cooperative units (two-state transitions)? (5) How do thermodynamic domains compare with molecular structures (if known) and from such a comparison, is there evidence for cooperativity in unfolding? Thermodynamic