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.
ABSTRACT
To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output (up to 50 mg/liter).
Subject(s)
Escherichia coli/genetics , Gene Expression , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Cloning, Molecular , Escherichia coli/metabolism , Humans , Multigene Family , Protein Conformation , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide-peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix-helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins.
Subject(s)
Bacterial Proteins/chemistry , Computer Simulation , DNA-Binding Proteins/chemistry , Models, Molecular , Receptor, EphA1/chemistry , Transcription Factors/chemistry , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Molecular Dynamics Simulation , Monte Carlo Method , Protein Multimerization , Protein Structure, Secondary , Receptor, EphA1/metabolism , Transcription Factors/genetics , beta-Galactosidase/chemistry , beta-Galactosidase/metabolismABSTRACT
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.
