M Boyer

Localization of a new α-actinin binding site in the COOH — terminal part of actin sequence

Summary The interaction of filamentous actin with α-actinin, an actin cross-linking protein, is well established. On the other hand, monomeric actin — α-actinin interaction has been a subject of controversy. In this report, we have characterized the interaction of monomeric actin, coated on plastic plates under conditions of non — polymerizatioon, with α-actinin in presence of magnesium. Using specific polyclonal anti — actin antibodies, with the whole molecule or purified peptides, we have localized two sites of interaction on actin module: one near Thr-103 and a new one in the twenty last amino acids.

Dynamic light scattering study of precrystallizing ribonuclease solutions

The translational diffusion coefficient of bovine pancreatic ribonuclease A, a globular protein, has been measured by dynamic light scattering under various conditions related to the crystallization process. In all cases, a monomodal light scattering autocorrelation function was observed. The diffusion coefficient exhibited a linear dependence on protein concentration and an interaction parameter could be calculated. Attractive interactions were found to increase with salt concentration and to decrease with pH in the presence of salts. Finally, optimal crystallization conditions correspond to moderate attractive interactions.

Protein interactions in concentrated ribonuclease solutions

To investigate the protein interactions involved in the crystallization process of ribonuclease A, dynamic light scattering (DLS) and small angle X-ray scattering experiments (SAXS) were performed on concentrated solutions. Whereas the translational diffusion coefficient obtained from DLS is sensitive to thermodynamic and hydrodynamic interactions and permits to calculate an interaction parameter, the shape of the SAXS curves is related to the type of interaction (attractive or repulsive). We compared the effect of pH on protein interactions in the case of two types of crystallizing agents: a mixture of salts (3 M sodium chloride plus 0.2 M ammonium sulfate) and an organic solvent (ethanol). The results show that in the presence of ethanol, as in low salt, protein interactions become more attractive as the pH increases from 4 to 8 and approaches the isoelectric point. In contrast, a reverse effect is observed in high salt conditions: the strength of attractive interactions decreases as the pH increases. The range of the pH effect can be related to ionization of histidine residues, particularly those located in the active site of the protein. The present observations point out the important role played by localized charges in crystallization conditions, whatever the precipitating agent.

Biochemical evidence for the presence of an unconventional actin protein in a prokaryotic organism

The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system. Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours. Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg(++)-ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin. This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment‐1 and two actin monomers

Two‐dimensional hydrophobic clusters analysis (HCA) was used to compare the distribution of hydrophobic clusters along various actin sequence. HCA‐deduced patterns were not altered by amino‐acid variations throughout the evolution of actin and we observed similar hydrophobic motifs comprising myosin subfragment‐1 ATP‐independent binding sites. HCA suggested the presence of two groups of identical hydrophobic motifs (A1 and A2) which bound on each side of the S1 (63 kDa‐31 kDa) connecting segment in relation with two actin monomers. This connection is important in communications between actin‐ and nucleotide‐binding sites. We postulate that some relation and message between the two motifs A1 and A2 take place through myosin subfragment‐1 (63 kDa‐31 kDa) connecting segment.

Crystallization of monoacylated proteins: influence of acyl chain length

Abstract The crystallization of monoacylated proteins has been investigated using a model system. Acylated derivatives of bovine pancreatic ribonuclease A, differing in their acyl chain lengths (10 to 16 carbon atoms), have been prepared using reverse micelles as microreactors. With one fatty acid moiety per polypeptide chain, covalently attached to the NH2 terminus of the protein, all the modified proteins have similar enzymatic activity and hydrodynamic radius as the native protein. Only the caprylated derivative can give crystals which diffract to high resolution. The resolved structure indicates that: (i) the protein folding is not modified by the chemical modification, (ii) the capryl moiety is not buried within the molecule but available for external interactions. Dynamic light scattering experiments on concentrated solutions show that protein-protein interactions are dependent on acyl chain length. Proteins with the longest attached chains (14 and 16 carbon atoms) tend to self-associate through acyl group interactions.