Conformational changes in actin-myosin isoforms probed by Ni(II).Gly-Gly-His reactivity.

Crucial information concerning conformational changes that occur during the mechanochemical cycle of actin-myosin complexes is lacking due to the difficulties encountered in obtaining their three-dimensional structures. To obtain such information, we employed a solution-based approach through the reaction of Ni(II).tripeptide chelates which are able to induce protein cleavage and cross-linking reactions. Three different myosin motor domain isoforms in the presence of actin and nucleotides were treated with a library of Ni(II).tripeptide chelates and two reactivities were observed: (1) muscle motor domains were cross-linked to actin, as also observed for the skeletal muscle isoform, while (2) the Dictyostelium discoideum motor domain was cleaved at a single locus. All Ni(II).tripeptide chelates tested generated identical reaction products, with Ni(II).Gly-Gly-His, containing a C-terminal carboxylate, exhibiting the highest reactivity. Mass spectrometric analysis showed that protein cleavage occurred within segment 242-265 of the Dictyostelium discoideum myosin heavy chain sequence, while the skeletal myosin cross-linking site was as localized previously within segment 506-561. Using a fusion protein consisting of the yellow and cyan variants of green fluorescent protein linked by Dictyostelium discoideum myosin segment 242-265, we demonstrated that the primary sequence of this segment alone is not a sufficient substrate for Ni(II).Gly-Gly-His-induced cleavage. Importantly, the cross-linking and cleavage reactions both exhibited specific structural sensitivities to the nature of the nucleotide bound to the active site, validating the conformational changes suggested from crystallographic data of the actin-free myosin motor domain.

Natural N-terminal fragments of brain abundant myristoylated protein BASP1.

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues. In this study, we show that BASP1 is accompanied by at least six BASP1 immunologically related proteins (BIRPs), which are present in all animal species studied (rat, bovine, human, chicken). BIRPs have lower molecular masses than that of BASP1. Similarly to BASP1, they are myristoylated. Peptide mapping and partial sequencing have shown that BIRPs represent a set of BASP1 N-terminal fragments devoid of C-terminal parts of different length. In a definite species, the same set of BASP1 fragments is present in both brain and other tissues. The sum amount of the fragments is about 50% of the BASP1 amount in a tissue. Obligatory accompanying of BASP1 by a set of specific fragments indicates that these fragments are of physiological significance.

Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit.

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.