Actin filament bundling and different nucleating effects of mouse Diaphanous-related formin FH2 domains on actin/ADF and actin/cofilin complexes.

Mouse Diaphanous-related formins (mDias) are members of the formin protein family that nucleate actin polymerization and subsequently promote filamentous actin (F-actin) elongation by monomer addition to fast-growing barbed ends. It has been suggested that mDias preferentially recruit actin complexed to profilin due to their proline-rich FH1 domains. During filament elongation, dimeric mDias remain attached to the barbed ends by their FH2 domains, which form an anti-parallel ring-like structure enclosing the filament barbed ends. Dimer formation of mDia-FH2 domains is dependent on their N-terminal lasso and linker subdomains (connector). Here, we investigated the effect of isolated FH2 domains on actin polymerization using mDia1-FH2 domain plus connector, as well as core mDia1, mDia2, and mDia3 missing the connector, by cosedimentation and electron microscopy after negative staining. Analytical ultracentrifugation showed that core FH2 domains of mDia1 and mDia2 exhibited a low degree of dimer formation, whereas mDia3-FH2 minus connector and mDia1-FH2 plus connector readily dimerized. Only core mDia3-FH2 was able to nucleate actin polymerization. However, all tested core FH2 domains decorated and bundled F-actin, as demonstrated by electron microscopy after negative staining. Bundling activity was highest for mDia3-FH2, decreased for mDia2-FH2, and further decreased for mDia1-FH2. The mDia1-FH2 domain plus connector induced actin polymerization also in the absence of profilin, but failed to induce F-actin deformation and bundling. We also tested whether mDia1-FH2 was able to repolymerize actin in complex with different proteins that stabilize globular actin. The data obtained demonstrated that mDia1-FH2 induced actin repolymerization only from the actin/cofilin-1 complex, but not when complexed to actin depolymerizing factor, gelsolin segment 1, vitamin D binding protein, or deoxyribonuclease I.

Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2).

The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.5 was characterized by intrinsic and extrinsic (Nile Red and 1,8-ANS) fluorescence spectroscopy, 90 degrees light-scattering and transmission electron microscopy (TEM). Compared to the pH 4.5 solution, rhBMP-2 at pH 6.5 had (i) a stronger intrinsic fluorescence intensity, (ii) a longer fluorescence lifetime, (iii) a stronger 90 degrees light-scattering intensity, (iv) a stronger Nile Red fluorescence intensity, (v) a higher Nile Red fluorescence anisotropy, (vi) a lower 1,8-ANS fluorescence intensity, (vii) a higher 1,8-ANS fluorescence anisotropy and (viii) a longer 1,8-ANS fluorescence lifetime. Electron microscopy showed that rhBMP-2 at pH 4.5 contained aggregates of about 100 nm in diameter. More and larger protein aggregates (0.1-2 microm) were observed in solution at pH 6.5. Taken together, these results indicate conformational changes and increased aggregation of rhBMP-2 at pH 6.5 compared to pH 4.5, demonstrating a strong influence of pH on rhBMP-2 physical stability. These observations must be considered when developing a delivery system for rhBMP-2.

Characterization of the head-to-tail overlap complexes formed by human lamin A, B1 and B2 "half-minilamin" dimers.

Half-minilamins, representing amino- and carboxy-terminal fragments of human lamins A, B1 and B2 with a truncated central rod domain, were investigated for their ability to form distinct head-to-tail-type dimer complexes. This mode of interaction represents an essential step in the longitudinal assembly reaction exhibited by full-length lamin dimers. As determined by analytical ultracentrifugation, the amino-terminal fragments were soluble under low ionic strength conditions sedimenting with distinct profiles and s-values (1.6-1.8 S) indicating the formation of coiled-coil dimers. The smaller carboxy-terminal fragments were, except for lamin B2, largely insoluble under these conditions. However, after equimolar amounts of homotypic amino- and carboxy-terminal lamin fragments had been mixed in 4 M urea, upon subsequent renaturation the carboxy-terminal fragments were completely rescued from precipitation and distinct soluble complexes with higher s-values (2.3-2.7 S) were obtained. From this behavior, we conclude that the amino- and carboxy-terminal coiled-coil dimers interact to form distinct oligomers (i.e. tetramers). Furthermore, a corresponding interaction occurred also between heterotypic pairs of A- and B-type lamin fragments. Hence, A-type lamin dimers may interact with B-type lamin dimers head-to-tail to yield linear polymers. These findings indicate that a lamin dimer principally has the freedom for a "combinatorial" head-to-tail association with all types of lamins, a property that might be of significant importance for the assembly of the nuclear lamina. Furthermore, we suggest that the head-to-tail interaction of the rod end domains represents a principal step in the assembly of cytoplasmic intermediate filament proteins too.

Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.

New methods allowing the detection of protein aggregates: a case study on trastuzumab.

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates.

Structure-based design of peptides that self-assemble into regular polyhedral nanoparticles.

Artificial particulate systems such as polymeric beads and liposomes are being applied in drug delivery, drug targeting, antigen display, vaccination, and other technologies. Here we used computer modeling to design a novel type of nanoparticles composed of peptides as building blocks. We verified the computer models via solid-phase peptide synthesis and biophysical analyses. We describe the structure-based design of a novel type of nanoparticles with regular polyhedral symmetry and a diameter of about 16 nm, which self-assembles from single polypeptide chains. Each peptide chain is composed of two coiled coil oligomerization domains with different oligomerization states joined by a short linker segment. In aqueous solution the peptides form nanoparticles of about 16 nm diameter. Such peptide nanoparticles are ideally suited for medical applications such as drug targeting and drug delivery systems, such as imaging devices, or they may be used for repetitive antigen display.

Myomesin is a molecular spring with adaptable elasticity.

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.

Specific binding of cinnamycin (Ro 09-0198) to phosphatidylethanolamine. Comparison between micellar and membrane environments.

Cinnamycin (Ro 09-0198) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE). Formation of a complex with phosphatidylethanolamine follows a 1:1 stoichiometry. Using high-sensitivity isothermal titration calorimetry (ITC), we have measured the thermodynamic parameters of complex formation for two different PE environments, namely, PE dissolved either in octyl glucoside (OG) micelles or in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer membrane. We have compared diacyl-PE with lyso-PE and have varied the carbon chain length from 6 to 18. Binding requires both a PE headgroup and at least one fatty acyl chain. The optimum chain length for complex formation (n) is eight. Longer chains do not enhance the binding affinity; for shorter chains, the interaction is weakened. The cinnamycin-PE complex has a binding constant K(0) of approximately 10(7)-10(8) M(-1) in the POPC membrane and only approximately 10(6) M(-1) in the octyl glucoside micelle. The difference can be attributed to the nonspecific hydrophobic interaction of cinnamycin with the lipid membrane. Complex formation is enthalpy-driven in OG micelles, whereas enthalpy and entropy make equal contributions in bilayer membranes. However, for the optimum chain length (n) of eight, the binding reaction is also completely enthalpy-driven for the bilayer membrane.

Specific binding of Ro 09-0198 (cinnamycin) to phosphatidylethanolamine: a thermodynamic analysis.

Ro 09-0198 (cinnamycin) is a tetracyclic peptide antibiotic that is used to monitor the transbilayer movement of phosphatidylethanolamine (PE) in biological membranes during cell division and apoptosis. The molecule is one of the very rare examples where a small peptide binds specifically to a particular lipid. In model membranes and biological membranes containing phosphatidylethanolamine, Ro 09-0198 forms a 1:1 complex with this lipid. We have measured the thermodynamic parameters of complex formation with high sensitivity isothermal titration calorimetry and have investigated the structural consequences with deuterium and phosphorus solid-state NMR. Complex formation is characterized by a large binding constant, K0, of 10(7) to 10(8) M(-1), depending on the experimental conditions. The reaction enthalpy, DeltaHdegrees, varies between zero at 10 degrees C to strongly exothermic -10 kcal/mol at 50 degrees C. For large vesicles with a diameter of approximately 100 nm, DeltaHdegrees decreases linearly with temperature and the molar heat capacity of complex formation can be evaluated as = -245 cal/mol, indicating a hydrophobic binding mechanism. The free energy of binding is DeltaGdegrees = -10.5 kcal/mol and shows only little temperature dependence. The constancy of DeltaGdegrees together with the distinct temperature-dependence of DeltaHdegrees provide evidence for an entropy-enthalpy compensation mechanism: at 10 degrees C, complex formation is completely entropy-driven, at 50 degrees C it is enthalpy-driven. Varying the PE fatty acid chain-length between 6 and 18 carbon atoms produces similar binding constants and DeltaHdegrees values. Addition of Ro 09-0198 to PE containing bilayers eliminates the typical bilayer structure and produces 2H- and 31P-NMR spectra characteristic of slow isotropic tumbling. This reorganization of the lipid matrix is not limited to PE but also includes other lipids.