[Show abstract][Hide abstract] ABSTRACT: Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) 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-β1m 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-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m 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-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m 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-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.
[Show abstract][Hide abstract] ABSTRACT: Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.
[Show abstract][Hide abstract] ABSTRACT: ErbB is a family of epidermal growth factor receptors representing an important class of receptor tyrosine kinases that play
a leading role in cellular growth, development, and differentiation. Transmembrane domains of these receptors transduce biochemical
signals across the plasma membrane via lateral homo- and heterodimerization. The relatively small size of ErbB transmembrane
domain complexes with detergents or lipids makes it possible to study their detailed spatial structure using three-dimensional
heteronuclear high-resolution NMR spectroscopy. Here, we describe an efficient expression system and a purification procedure
for preparative-scale production of transmembrane peptides from all four ErbB proteins—ErbB1, ErbB2, ErbB3, and ErbB4—for
the purpose of structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS cells as N-terminal extensions of thioredoxin A. The fusion proteins were cleaved with the light chain of
human enterokinase. Several (10–30) milligrams of purified isotope-labeled transmembrane peptides were isolated using a simple
and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange
chromatography. The purified peptides were reconstituted in a lipid/detergent environment (micelles or bicelles) and characterized
using dynamic light scattering and CD and NMR spectroscopy. The data obtained indicate that purified ErbB transmembrane peptides
are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.
Keywordsmembrane protein–ErbB–bacterial expression–purification–detergent solubilization–NMR
[Show abstract][Hide abstract] ABSTRACT: Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.
Acta Naturae 04/2011; 3(2):90-8. · 1.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.
[Show abstract][Hide abstract] ABSTRACT: The structures of two crystal modifications of the W34F mutant ribonuclease from the bacterium Bacillus intermedius (binase) were solved and refined at 1.7 and 1.1 A resolution. The kinetic parameters of the hydrolysis of substrates of different lengths (GpU, GpUp, and poly(I)) by binase and its W34F mutant were investigated and compared. The catalytic activity of the enzymes was shown to increase with increasing length of the substrate. The substitution of tryptophan for phenylalanine does not lead to a change in the activity of the enzyme but results in a decrease in the binding constants for substrates containing more than one phosphate groups. A comparison of the structure of the mutant enzyme with the previously established structures of binase and its complexes with sulfate ions and guanosine monophosphate showed that the difference in their kinetic parameters is related to the fact that the mutant ribonuclease cannot bind the second phosphate group. Both crystal modifications of the mutant binase contain dimers, like in the crystal structure of binase studied previously. In these dimers, only one enzyme molecule can bind the substrate molecule. Since the dimers were found in the crystals grown under four different conditions, it can be suggested that the enzyme can exist as dimers in solution as well. Mutants of binase, which could exclude the formation of dimers, are suggested.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of the American Chemical Society 07/2009; 131(24):8564-70. DOI:10.1021/ja901602c · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.