The architecture of transmembrane and cytoplasmic juxtamembrane regions of Toll-like receptors.

Toll-like receptors (TLRs) are the important participants of the innate immune response. Their spatial organization is well studied for the ligand-binding domains, while a lot of questions remain unanswered for the membrane and cytoplasmic regions of the proteins. Here we use solution NMR spectroscopy and computer simulations to investigate the spatial structures of transmembrane and cytoplasmic juxtamembrane regions of TLR2, TLR3, TLR5, and TLR9. According to our data, all the proteins reveal the presence of a previously unreported structural element, the cytoplasmic hydrophobic juxtamembrane α-helix. As indicated by the functional tests in living cells and bioinformatic analysis, this helix is important for receptor activation and plays a role, more complicated than a linker, connecting the transmembrane and cytoplasmic parts of the proteins.

Expression, purification and characterization of SORCS2 intracellular domain for structural studies.

Neurotrophin signaling pathways are one of the major cascades in neuronal development and involved in many key processes including proliferation, differentiation, apoptosis, synaptic plasticity, axonal growth. In addition to the main classes of neurotrophin receptors, Trk and P75NTR, there are many auxiliary proteins, which can also bind neurotrophins and regulate the signaling pathways. The versatility of interactions between them could explain multiple and completely opposite biological outcomes such as cell survival or apoptosis. Membrane protein SorCS2, a vacuolar protein sorting 10 protein-domain receptor, interacts with P75NTR and controls the activity of Trk receptors. The abnormal functioning of SorCS2 is associated with neurodegenerative diseases, such as Alzheimer's and Huntington's disease. But the mechanism of SorCS2 activation and basis of the interaction with P75NTR has remained elusive. Herein, we describe two efficient approaches for the intracellular domain of the SorCS2 production employing bacterial and cell-free expression systems, as well as purification and refolding protocols. Finally, we characterized the purified protein by DLS and NMR and demonstrated that the protein sample is suitable for structural studies.

The Recombinant Luciferase of the Fungus Neonothopanus nambi: Obtaining and Properties.

A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.

Characterization of Small Isotropic Bicelles with Various Compositions.

Structural studies of membrane proteins are of great importance and interest, with solution and solid state NMR spectroscopy being very promising tools for that task. However, such investigations are hindered by a number of obstacles, and in the first place by the fact that membrane proteins need an adequate environment that models the cell membrane. One of the most widely used and prospective membrane mimetics is isotropic bicelles. While large anisotropic bicelles are well-studied, the field of small bicelles contains a lot of "white spots". The present work reports the radii of particles and concentration of the detergents in the monomeric state in solutions of isotropic bicelles, formed by 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and sodium cholate, as a function of lipid/detergent ratio and temperature. These parameters were measured using (1)H NMR diffusion spectroscopy for the bicelles composed of lipids with saturated fatty chains of different length and lipids, containing unsaturated fatty acid residue. The influence of a model transmembrane protein (membrane domain of rat TrkA) on the properties of bicelles and the effect of the bicelle size and composition on the properties of the transmembrane protein were investigated with heteronuclear NMR and nuclear Overhauser effect spectroscopy. We show that isotropic bicelles that are applicable for solution NMR spectroscopy behave as predicted by the theoretical models and are likely to be bicelles rather than mixed micelles. Using the obtained data, we propose a simple approach to control the size of bicelles at low concentrations. On the basis of our results, we compared different rim-forming agents and selected CHAPS as a detergent of choice for structural studies in bicelles, if the deuteration of the detergent is not required.

[Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies].

Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

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.

Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment.

The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their(15)N- and [(15)N,(13)C]-isotope-labelled derivatives, were described. Each peptide was produced inEscherichia coliBL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.

Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels.

An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives ((15)N-, (15)N-/13C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly alpha-helical conformation. The existence of cross-peaks for all glycines of the (15)N-HSQC NMR spectra as well as relatively small line widths (~20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.