The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) MgCl2/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/20 ,tM GTP (buffer A), disrupted in a glass homogenizer, and centrifuged at 640 x g for 15 min, and the supernatant was decanted. The residue was twice resuspended in 70 ml of buffer A and recentrifuged. The combined supernatants containing the cell membrane fragments were centrifuged at 186,000 x g for 90 min, and the pellet was resuspended in 50 ml of buffer A containing 2% octyl glucoside, homogenized, and gently shaken for 45 min. The solution was centrifuged at 142,000 x g for 90 min, and the supematant was filtered through a 0.45-,tm Minisart cellulose acetate filter (Sartorius). The solution (-0.4 mg of protein per ml) was loaded into a 10-ml MonoS column equili-

Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.

This work shows that the partial replacement of diamagnetic Ca 2+ by paramagnetic Tb 3+ in Ca 2+ /calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb 3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca 2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb 3+ ions bound to the other domain at distances of over 40 A î from the Tb 3+ ion, shifting the resonances for these nuclei. In addition, the Tb 3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15 N-1 H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb 3+ ions. Pseudocontact shifts for NH groups and 1 H-15 N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.z 1999 Federation of European Biochemical Societies.

No abstract

The cytoskeleton of mammalian cells consists of families of protein fibers and networks which include microtubules (MT), microfilaments (MF), clathrin baskets (CB), intermediate filaments (IF), and the microtrabeculae lattice (MTL). The networks function in a coordinated, integrated manner to bring about movement/motility of intracellular organelle and membrane traffic. Although much is known about the structure and function of MT and MF, more knowledge is needed to elucidate the ultrastructure, assembly and function of intermediate filaments in non-keratinized mammalian cells.The objective of this study was to elucidate the ultrastructure of intermediate filaments isolated and purified from cultured mammalian cells.Macrophages were harvested from the peritoneal cavities of mice. Triton- insoluble fractions of IF were isolated from spread cultures using high salt and low ionic strength buffers. Purified IF were analyzed biochemically. Using poly acrylamide gel electrophoresis IF consists of 4-7 major bands in the molecular weight ranges of 14-90Kd3.