Membrane Proteins In Lipid Environments Research Articles

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments.

Although membrane proteins are in the focus of biochemical research for many decades the general knowledge of this important class is far behind soluble proteins. Despite several recent technical developments, the most challenging feature still is the generation of high-quality samples in environments suitable for the selected application. Reconstitution of membrane proteins into lipid bilayers will generate the most native-like environment and is therefore commonly desired. However, it poses tremendous problems to solution-state NMR analysis due to the dramatic increase in particle size resulting in high rotational correlation times. Nevertheless, a few promising strategies for the solution NMR analysis of membrane inserted proteins are emerging and will be discussed in this chapter. We focus on the generation of membrane protein samples in nanodisc membranes by cell-free systems and will describe the characteristic advantages of that platform in providing tailored protein expression and folding environments. We indicate frequent problems that have to be overcome in cell-free synthesis, nanodisc preparation, and customization for samples dedicated for solution-state NMR. Detailed instructions for sample preparation are given, and solution NMR approaches suitable for membrane proteins in bilayers are compiled. We further discuss the current strategies applied for signal detection from such difficult samples and describe the type of information that can be extracted from the various experiments. In summary, a comprehensive guideline for the analysis of membrane proteins in native-like membrane environments by solution-state NMR techniques will be provided.

Proton-Based Structural Analysis of a Heptahelical Transmembrane Protein in Lipid Bilayers.

The structures and properties of membrane proteins in lipid bilayers are expected to closely resemble those in native cell-membrane environments, although they have been difficult to elucidate. By performing solid-state NMR measurements at very fast (100 kHz) magic-angle spinning rates and at high (23.5 T) magnetic field, severe sensitivity and resolution challenges are overcome, enabling the atomic-level characterization of membrane proteins in lipid environments. This is demonstrated by extensive 1H-based resonance assignments of the fully protonated heptahelical membrane protein proteorhodopsin, and the efficient identification of numerous 1H–1H dipolar interactions, which provide distance constraints, inter-residue proximities, relative orientations of secondary structural elements, and protein–cofactor interactions in the hydrophobic transmembrane regions. These results establish a general approach for high-resolution structural studies of membrane proteins in lipid environments via solid-state NMR.

C3-O-04Functional structures of ion channels in lipid environment

The structural study of membrane protein is getting more and more popular as for the importance in its function especially for drug discovery. Recent progress in structure and function relationship of membrane proteins is based on the near atomic structures obtained by X-ray crystallography. Recently, cryo-EM single particle reconstruction of membrane proteins has been reported in such a resolution range due to the benefit of direct electron detector and high performance computation [ 1 , 2 ]. Even though those are useful to understand the structure and function relationship, it would be important to remind that there are in detergent solubilized state. The final goal of structural study of membrane protein is to utilize the membrane proteins in lipid environment. We have developed alternative methods so-called Random Spherically-Constrained (RSC) reconstruction in which membrane proteins on liposome is directly imaged in cryo-EM [ 3 ]. We have struggled a lot to optimize sample preparation, specimen preparation, imaging condition and image processing algorithm and then, now we have achieved sub-nanometer resolution reconstruction of potassium channel, Kv1.2-beta2.1 in detergent free, lipid environment.

A knowledge‐based potential highlights unique features of membrane α‐helical and β‐barrel protein insertion and folding

Outer membrane β-barrel proteins differ from α-helical inner membrane proteins in lipid environment, secondary structure, and the proposed processes of folding and insertion. It is reasonable to expect that outer membrane proteins may contain primary sequence information specific for their folding and insertion behavior. In previous work, a depth-dependent insertion potential, E(z) , was derived for α-helical inner membrane proteins. We have generated an equivalent potential for TM β-barrel proteins. The similarities and differences between these two potentials provide insight into unique aspects of the folding and insertion of β-barrel membrane proteins. This potential can predict orientation within the membrane and identify functional residues involved in intermolecular interactions.

GRIFFIN: A versatile methodology for optimization of protein-lipid interfaces for membrane protein simulations.

As new atomic structures of membrane proteins are resolved, they reveal increasingly complex transmembrane topologies, and highly irregular surfaces with crevices and pores. In many cases, specific interactions formed with the lipid membrane are functionally crucial, as is the overall lipid composition. Compounded with increasing protein size, these characteristics pose a challenge for the construction of simulation models of membrane proteins in lipid environments; clearly, that these models are sufficiently realistic bears upon the reliability of simulation-based studies of these systems. Here, we introduce GRIFFIN, which uses a versatile framework to automate and improve a widely-used membrane-embedding protocol. Initially, GRIFFIN carves out lipid and water molecules from a volume equivalent to that of the protein, so as to conserve the system density. In the subsequent optimization phase GRIFFIN adds an implicit grid-based protein force-field to a molecular dynamics simulation of the pre-carved membrane. In this force-field, atoms inside the implicit protein volume experience an outward force that will expel them from that volume, whereas those outside are subject to electrostatic and van-der-Waals interactions with the implicit protein. At each step of the simulation, these forces are updated by GRIFFIN and combined with the intermolecular forces of the explicit lipid-water system. This procedure enables the construction of realistic and reproducible starting configurations of the protein-membrane interface within a reasonable timeframe and with minimal intervention. GRIFFIN is a standalone tool designed to work alongside any existing molecular dynamics package, such as NAMD or GROMACS.

The thermodynamics of simple biomembrane mimetic systems.

Insight into the forces governing a system is essential for understanding its behavior and function. Thermodynamic investigations provide a wealth of information that is not, or is hardly, available from other methods. This article reviews thermodynamic approaches and assays to measure collective properties such as heat adsorption / emission and volume variations. These methods can be successfully applied to the study of lipid vesicles (liposomes) and biological membranes. With respect to instrumentation, differential scanning calorimetry, pressure perturbation calorimetry, isothermal titration calorimetry, dilatometry, and acoustic techniques aimed at measuring the isothermal and adiabatic processes, two- and three-dimensional compressibilities are considered. Applications of these techniques to lipid systems include the measurement of different thermodynamic parameters and a detailed characterization of thermotropic, barotropic, and lyotropic phase behavior. The membrane binding and / or partitioning of solutes (proteins, peptides, drugs, surfactants, ions, etc.) can also be quantified and modeled. Many thermodynamic assays are available for studying the effect of proteins and other additives on membranes, characterizing non-ideal mixing, domain formation, bilayer stability, curvature strain, permeability, solubilization, and fusion. Studies of membrane proteins in lipid environments elucidate lipid–protein interactions in membranes. Finally, a plethora of relaxation phenomena toward equilibrium thermodynamic structures can be also investigated. The systems are described in terms of enthalpic and entropic forces, equilibrium constants, heat capacities, partial volume changes, volume and area compressibility, and so on, also shedding light on the stability of the structures and the molecular origin and mechanism of the structural changes.

Structure determination of membrane proteins by NMR spectroscopy

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.

14] Nuclear magnetic resonance of membrane-associated peptides and proteins

Structural biology is based on the premise that the fundamental understanding of biological functions lies in the three-dimensional structures of proteins and other biopolymers. The two well-established experimental methods for determining the structures of proteins work very well for globular proteins: witness the explosive growth of the Protein Data Bank (PDB). However, approximately 30% of all expressed polypeptides are membrane-associated, and neither X-ray crystallography nor solution nuclear magnetic resonance (NMR) spectroscopy is very effective for these proteins. The lipids required for the structural integrity and functionality of membrane proteins impede crystallization as well as the rate of overall reorientation in solution. NMR of Proteins NMR spectroscopy can be applied to wide variety of samples, ranging from isotropic solutions to crystalline powders, including those with slowly reorienting or immobile macromolecules, such as membrane proteins in lipid environments. NMR is capable of resolving signals from all atomic sites in proteins, and each site has several well-characterized nuclear spin interactions that can be used as sources of information about molecular structure and dynamics, as well as chemical interactions. The spin interactions can be probed through radio frequency (rf) irradiations and sample manipulations that lead to complementary strategies for NMR spectroscopy of membrane proteins reconstituted in lipid micelles or bilayers. Comparisons between the results obtained with solution NMR experiments on lipid micelle samples, and solid-state NMR experiments on lipid bilayer samples, are especially valuable for membrane proteins with predominantly helical secondary structure. Multidimensional solution NMR methods can be successfully applied to relatively small membrane proteins in micelles; however, the size limitation is substantially more severe than for globular proteins because the many lipid molecules associated with each polypeptide slow its overall reorientation rate. In particular, using currently available instruments and methods, it is difficult to resolve, assign, and measure the “long-range” nuclear overhauser effects (NOEs) between hydrogens on hydrophobic side-chains that are needed to determine tertiary structures based on distance constraints. However, the ability to weakly align membrane proteins in micelles enables the measurement of residual dipolar couplings, and improves the feasibility of determining the structures of membrane proteins using solution NMR methods. Nonetheless, it is highly desirable to determine the structures of membrane proteins in the definitive environment of phospholipid bilayers, where solution NMR methods fail completely for all classes of membrane proteins. Fortunately, solid-state NMR spectroscopy is well suited for peptides and proteins immobilized in phospholipid bilayers. Both oriented sample and magic angle spinning methods provide approaches to measuring orientational and distance parameters for structure determination.