Targeting Of Integral Membrane Proteins Research Articles

Bioelectronic Measurement of Target Engagement to a Membrane-Bound Transporter.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable. However, the mass dependence of SPR signals may preclude the detection of binding events when the ligand has a significantly smaller mass than the target protein. In FRET-based experiments, protein labeling through modification may inadvertently alter protein dynamics. Graphene Bio-Electronic Sensing Technology (GBEST) aims to overcome these challenges. Label-free characterization takes place in a microfluidic chamber wherein a fluid lipid membrane is reconstituted directly above the GBEST sensor surface. By leveraging the high conductivity, sensitivity, and electrical properties of monolayer graphene, minute changes in electrostatic charges arising from the binding and unbinding of a ligand to a native IMP target can be detected in real time and in a mass-independent manner. Using crude membrane fractions prepared from cells overexpressing monocarboxylate transporter 1 (MCT1), we demonstrate the ability to (1) form a fluid lipid bilayer enriched with MCT1 directly on top of the GBEST sensor and (2) obtain kinetic binding data for an anti-MCT1 antibody. Further development of this novel technology will enable characterization of target engagement by both low- and high-molecular-weight drug candidates to native IMP targets in a physiologically relevant membrane environment.

Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance

BackgroundThe E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts.ResultsUsing GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins.ConclusionsWe demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.

Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. The protocol steps include generation of viruses, virus quality control, and initial expression trials utilizing standard commercial baculovirus vector systems and are exemplified for G protein-coupled receptor targets. The viral quality, quantity, and recombinant protein expression are evaluated by microscopy, flow cytometry, fluorimetry, and SDS-PAGE, using either covalently fused fluorescent proteins or co-expressed fluorescence markers. Moreover, integral membrane protein expression levels, approximate molecular mass, and stability can be evaluated from small-scale expression and purification trials.

Development of a biochemical and biophysical suite for integral membrane protein targets: A review

The generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug design (SBDD) in drug discovery projects. We furthermore include timeline estimates and an overview of the target selection and biophysical screening approaches.

The cubicon method for concentrating membrane proteins in the cubic mesophase.

The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible-using traditional methods-to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3-5 h.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

EHD3 Mediates Integral Membrane Protein Trafficking and Maintain Electrical Excitability and Adrenergic Responsiveness in the Heart

Cardiac contraction relies upon the proper expression, trafficking, and retention of integral membrane proteins such as ion channels, transporters, and hormone receptors. These proteins govern cardiac contraction and short and long term adaptations to physiological and pathophysiological stimuli. The profile of expressed proteins is dynamic, and it is regulated to assure the proper response to stress. Yet, despite its obvious importance, little is known regarding even the identity (or the function) of the intracellular molecular pathways underpinning the trafficking and targeting of integral membrane proteins in the context of the native heart. The goal of this work is identify new molecular players that regulate protein targeting and trafficking in the heart. Eps 15 homology domain-containing (EHD) gene products (EHD1-4) are intracellular proteins that are key regulators of endosomal trafficking, lipid homeostasis, membrane protein recycling and trafficking. Previously uncharacterized in the heart, we recently presented evidence which demonstrated that this protein family likely plays indispensable roles in protein trafficking in cardiac muscle. Notably, an essential role for one of these proteins, EHD3, in the membrane trafficking of the Na/Ca exchanger (NCX) in heart was uncovered. The goal of this research program is for the first time to directly test the role of these proteins in cardiac structural and electrical activity using cutting-edge in vivo models of EHD deficiency. We show that EHD3 deficiency in the heart leads to: 1) abnormal cardiac structure at baseline; 2) irregular action potential morphology, heart rhythm, and conduction; 3) depressed β-adrenergic responsiveness; 4) dysfunctional NCX and LTCC trafficking and function; and 5) dysregulated ankyrin-B expression and trafficking. These data strongly support a role for EHD3 in membrane protein trafficking and regulation within the context of the native heart.

The N-terminal hydrophobic segment of Streptomyces coelicolor FtsY forms a transmembrane structure to stabilize its membrane localization

FtsY is the receptor of the signal recognition particle that mediates the targeting of integral membrane proteins in bacteria. It was shown that in Escherichia coli, the N-terminal region of FtsY contributes to its interaction with the membrane, but it is not inserted into the membrane. However, this study presents evidence that in Streptomyces coelicolor, FtsY has a hydrophobic region at its N-terminus, which forms a membrane insertion structure and contributes significantly to the binding between FtsY and membrane. Through membrane protein extraction followed by immunoblotting, we demonstrated that deletion of the N-terminal residues 11-39 from the S. coelicolor FtsY (ScFtsY) drastically reduced its membrane-binding capability and that the N-terminus of ScFtsY alone was capable of targeting the soluble EGFP protein onto the membrane with high efficiency. Furthermore, in a labeling experiment with the membrane-impermeable probe Mal-PEG, the ScFtsY N-terminal region was protected by the membrane and was not labeled. This observation indicates that this region was inserted into the membrane.

Self-Assembled Nanometer Scale Lipid Bilayers for Elucidating the Structure and Function of Membrane Proteins

One of the most challenging problems in biochemical science are studies of integral membrane proteins. Using designed amphipathic helical peptides, termed membrane scaffold proteins (MSPs) we have developed a simple and robust system that self-assembles phospholipids into soluble discoidal phospholipid bilayers 8-10 nm in diameter, termed Nanodiscs. Importantly, the same self-assembly process can be used to directly incorporate a variety of integral membrane proteins into these nanobilayers. The result is a native-like environment that provides stability and full functionality for these integral membrane protein targets. Using MSPs of varying lengths, the size and composition of the Nanodiscs can also control the oligomerization state of incorporated targets. During my presentation I will relay our most recent results in the incorporation of integral membrane enzymes, channels, transporters and receptors into Nanodiscs as well as the structural, biophysical and biochemical characterization of these nanostructures. We have also demonstrated that this same self-assembly process can be used to form Soluble Membrane Protein Libraries (SMPL) which can be used as the basis for high-throughput screening of protein-protein, cell-cell and cell-viral interactions. Supported by grants from the National Institutes of Health.

Truncated Isoforms of Kap60 Facilitate Trafficking of Heh2 to the Nuclear Envelope

Isoforms of importin-α have been identified in insect and human cells, and cross-linking experiments suggest that at least one isoform in each species participates in the targeting of integral membrane proteins to the inner nuclear membrane (INM). To directly test this hypothesis, an assay was developed using Saccharomyces cerevisiae. The data show that internal promoters are present within KAP60, and the nested transcripts are translated into three isoforms: Kap60-44, Kap60-30 and Kap60-10. In the absence of the isoforms, the INM protein Heh2-green fluorescent protein (GFP) localized to cytoplasmic membranes, whereas its wild-type localization at the nuclear periphery was restored when the Kap60-44 isoform was reintroduced. An INM-sorting sequence has been identified that cross-links with the isoform of importin-α that directs trafficking toward the nuclear envelope (NE). When this sequence in HEH2 was mutated, Heh2 was again localized to cytoplasmic membranes. Thus, this report provides the first evidence that isoforms of Kap60 exist in yeast, and these isoforms participate in the molecular sorting and enrichment of INM proteins to the NE. Herein, we provide additional support for the hypothesis that trafficking of INM proteins to the NE is a regulated process.

Functional substitution of the transient membrane-anchor domain inEscherichia coliFtsY with an N-terminal hydrophobic segment ofStreptomyces lividansFtsY

FtsY is a signal recognition particle receptor in Escherichia coli that mediates the targeting of integral membrane proteins to translocons by interacting with both signal recognition particle (SRP)-nascent polypeptide-ribosome complexes and the cytoplasmic membrane. Genes encoding the N-terminal segments of Streptomyces lividans FtsY were fused to a gene encoding the E. coli FtsY NG domain (truncated versions of FtsY lacking the transient membrane-anchor domain at the N-terminus), introduced into a conditional ftsY-deletion mutant of E. coli, and expressed in trans to produce chimeric FtsY proteins. Under FtsY-depleted conditions, strains producing chimeric proteins including 34 N-terminal hydrophobic residues grew whereas strains producing chimeric proteins without these 34 residues did not. A strain producing the chimeric protein comprising the 34 residues and NG domain processed beta-lactamase, suggesting that the SRP-dependent membrane integration of leader peptidase was restored in this strain. These results suggest that the N-terminal hydrophobic segment of FtsY in this Gram-positive bacterium is responsible for its interaction with the cytoplasmic membrane.

Proteomics of Synechocystis sp. PCC 6803

The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI‐TOF MS. More than half of the proteins were predicted to be integral with 1–12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.

Sorting of membrane proteins to vacuoles in plant cells

Plant cells may generate and maintain at least two separate vacuolar organelles, storage vacuoles and lytic vacuoles. In contrast to the relatively well-studied vacuolar sorting mechanisms for some soluble proteins in plant cells, little is known about the mechanisms of membrane protein sorting to these separate organelles, a process which is facilitated by transport vesicles and requires targeting information within the integral membrane proteins. During the past few years, the identification of a sorting receptor, an integral membrane protein for the lytic vacuole pathway, and the purification and characterization of novel dense vesicles that traffic to protein storage vacuoles in plant cells have provided new markers for studying the traffic of membrane proteins to vacuoles. Here we discuss recent progress in biochemical studies of vacuolar targeting of integral membrane proteins and immunocytochemical characterization of these newly identified transport vesicles. Possible models of sorting mechanisms in plant cells will be presented.

Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.