New strategy for high-level expression and purification of biologically active monomeric TGF-ß1/C77S in Escherichia coli.

Mature transforming growth factor beta1 (TGF-ß1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-ß1/C77S (further termed TGF-ß1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-ß1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-ß1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-ß1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-ß1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-ß1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-ß1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

Left-handed dimer of EphA2 transmembrane domain: Helix packing diversity among receptor tyrosine kinases.

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.

Simultaneous measurement of residual dipolar couplings for proteins in complex using the isotopically discriminated NMR approach.

One-bond residual dipolar couplings (RDCs) measured for the amide groups of proteins partially aligned in a magnetic field provide valuable information regarding the relative orientation of protein units. In order for RDCs obtained for individual proteins to be useful in the structure determination of heterodimer complexes, they should be measured for exactly the same alignment of the complex. Here, an isotopically discriminated IDIS-RDC-TROSY NMR experiment is proposed, which enables the measurement of HN RDCs for two proteins simultaneously and independently, but in the same sample, while they are part of the same complex. The signals for both proteins, one of which should be labeled with (15)N and the other with (15)N and (13)C, are observed in different subspectra, thus reducing spectral overlap. The approach uniquely ensures that RDCs measured for both proteins relate to exactly the same alignment tensor, allowing accurate measurement of the relative angle between the two proteins. The method is also applicable for complexes containing three or more protein components. The experiment can speed up and lead to automation of protein-protein docking on the basis of angular restraints.

Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state.

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

Determination of protein rotational correlation time from NMR relaxation data at various solvent viscosities.

An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR. In the present work, an innovative approach to the determination of the protein rotational correlation time tau(R) from the heteronuclear relaxation data is proposed. The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution. The method has been tested on computer simulated relaxation data as compared to the traditional tau(R) determination method from T(1)/T(2) ratio. The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component. The resulting rotational correlation time of 5.56 +/- 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T(1)/T(2) ratio.