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1.
Integral membrane proteins (IMPs) are difficult to identify, mainly for two reasons: the hydrophobicity of IMPs and their low abundance. Sample preparation is a key component in the large-scale identification of IMPs. In this review, we survey strategies for shotgun identification of IMPs by MS/MS. We will discuss enrichment, solubilization, separation, and digestion of IMPs, and data analysis for membrane proteomics. 相似文献
2.
Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large
macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology
has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes
to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed
by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest
to the biotechnology community. 相似文献
3.
Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11
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Melissa L. Fowler Jacob A. McPhail Meredith L. Jenkins Glenn R. Masson Florentine U. Rutaganira Kevan M. Shokat Roger L. Williams John E. Burke 《Protein science : a publication of the Protein Society》2016,25(4):826-839
The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches. 相似文献
4.
5.
Aims: To investigate whether Vibrio parahaemolyticus can sense and directly respond to the presence of cationic antimicrobial peptides (AMPs). Methods and Results: We performed proteomic methodologies to investigate the responsive proteins of V. parahaemolyticus on exposure to AMP Q6. Differential expression patterns of the outer membrane, inner membrane and cytoplasmic proteins (OMPs, IMPs and CPs) from the bacteria with or without Q6 treatment were obtained using two‐dimensional gel electrophoresis (2‐DE). Three OMPs (maltoporin, flagellin and OmpV), two IMPs (ATP synthase F1, alpha subunit; and OmpV) and three CPs (pyruvate dehydrogenase subunit E1, glyceraldehyde‐3‐phosphate dehydrogenase and inositol‐5‐monophosphate dehydrogenase) were identified using LC‐ESI‐Q‐TOF MS/MS and Mascot program. Real‐time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. Conclusions: Our results suggested that V. parahaemolyticus may directly respond to AMPs through the upregulation of the efflux channel, increased yield of energy, effective repair of damaged membranes and downregulation of carbohydrate and nucleotide metabolism for energy preservation. Significance and Impact of the Study: AMP‐responsive proteins identified in the current study could serve as attractive targets for developing more effective antimicrobial agents. 相似文献
6.
Claudia S. Kielkopf Madhubrata Ghosh Ganesh S. Anand Simon H.J. Brown 《Protein science : a publication of the Protein Society》2019,28(2):365-374
Apolipoprotein‐D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein‐D adopts an eight‐stranded β‐barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α‐helix. Crystallography studies of recombinant apolipoprotein‐D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX‐MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X‐ray crystallography, making HDX‐MS an invaluable orthogonal technique. Here, we report an HDX‐MS protocol for apolipoprotein‐D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand‐free apolipoprotein‐D revealed apolipoprotein‐D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein‐D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N‐terminus and C‐terminus. Together, our experiments provide insight into apolipoprotein‐D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein‐D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein‐D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein‐D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein‐D structure from X‐ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein‐D, to elucidate how proteins change flexibility when binding to small molecules. 相似文献
7.
Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified. 相似文献
8.
Mathias RA Chen YS Kapp EA Greening DW Mathivanan S Simpson RJ 《Methods (San Diego, Calif.)》2011,54(4):396-406
Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC–MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC–MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD. 相似文献
9.
Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia. 相似文献
10.
Xiaoxuan Lin Adam M. Zmyslowski Isabelle A. Gagnon Robert K. Nakamoto Tobin R. Sosnick 《Protein science : a publication of the Protein Society》2022,31(9)
Hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX‐MS in living Escherichia coli cells applied to BtuB, a TonB‐dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX‐MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX‐MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native‐like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB''s plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 binding‐induced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX‐MS studies in a native cellular environment.IMPORTANCEWe present a protocol for performing in vivo HDX‐MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically important for B12 transport. The in vivo HDX‐MS data corroborate the conclusions from our previous in vitro HDX‐MS study of the allostery initiated by B12 binding. Our success with BtuB and other proteins opens the possibility for performing additional HDX‐MS studies in a native cellular environment. 相似文献
11.
The analysis of integral membrane proteins (IMPs) with mass spectrometry-centered technologies has undergone great progress during the past few years, allowing for the analysis of several hundreds of IMPs. In this study, we investigated three promising shotgun approaches for the identification of IMPs of the model organism Bacillus subtilis. One comprises a classical membrane preparation procedure with carbonate and high-ionic-strength buffers, followed by SDS-PAGE and LC-MS/MS analysis. The two others are based on enzymatic trimming of the crude membrane fraction either with trypsin or proteinase K and subsequent gel-free analysis. As a result, we observed the highest degree of complementarity between the gel-based and the proteinase K approach, since the first exclusively addresses soluble loops and domains of IMPs and gave rise to 8709 unique peptides, whereas the latter contributed 1180 unique peptide identifications from otherwise inaccessible transmembrane helices (TMHs). All three methods contribute significant numbers (381, 284, and 276, respectively) to the total of 527 IMP identifications from the membrane fraction of exponentially growing B. subtilis cells, thus representing approximately 69% of all transcribed IMPs. 相似文献
12.
Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB. 相似文献
13.
Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs. 相似文献
14.
Alpha-helical integral-membrane proteins (IMPs) play a key role in many biological processes, such as signal transduction, and are targets for >50% of current therapeutic drugs. In contrast to their significant abundance and biological importance, they comprise <1% of structurally solved proteins. In the absence of experimental evidence, molecular modeling of IMP structures is an alternative for providing structural information and aiding further experimental design. In the current work, we propose two new amino acid lipid-facing propensity scales derived from the structural analysis of a nonredundant set of water-soluble proteins. The new scales, pi and delta, perform as well or better than published scales (Carugo's hydrophobicity and kPROT scales) in predicting the lipid-facing side of helical segments of a set of structurally solved IMPs, thus indicating (a) that the folding properties of water-soluble proteins and IMPs are similar, and (b) that the new scales will prove useful in modeling the transmembrane segments of IMPs. 相似文献
15.
Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct 总被引:1,自引:0,他引:1
Yu MJ Pisitkun T Wang G Aranda JF Gonzales PA Tchapyjnikov D Shen RF Alonso MA Knepper MA 《American journal of physiology. Cell physiology》2008,295(3):C661-C678
In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane "raft" proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n = 22) or immunoblotting (n = 6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action. 相似文献
16.
New insights into interactions between the nucleotide‐binding domain of CFTR and keratin 8
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Arkadiusz Bonna Grazyna Faure Tomasz Fraczyk Ariel Roldan Brice Hoffmann Mélanie Faria da Cunha Harald Herrmann Gergely L. Lukacs Aleksander Edelman Michał Dadlez 《Protein science : a publication of the Protein Society》2017,26(2):343-354
The intermediate filament protein keratin 8 (K8) interacts with the nucleotide‐binding domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508‐CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for CF patients bearing the ΔF508 mutation. Here, we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX‐MS) on recombinant wild‐type (wt) NBD1 and ΔF508‐NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt‐NBD1 and ΔF508‐NBD1. Finally, we performed HDX‐MS analysis of the NBD1 molecules and full‐length K8, revealing hydrogen‐bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508‐NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1. 相似文献
17.
Integral membrane proteins (IMPs) are popular target for drugs, but their resolved structures have been overlooked when compared with cytosolic proteins. The main reason is that IMPs usually need intensive post-translational modifications and they are bound to membranes, which increase the complexity of purifying or crystalizing them. Although different expression systems are used to express IMPs, baculovirus is considered one of the most successful expression systems for those proteins. Despite that, there are always unknown discrepancies in the level of IMPs expression in the baculovirus expression system. Retrospective studies have shown that expression of an immunoglobulin (anti-Chymase mouse monoclonal IgG1) driven by vp39 promoter was more efficient compared to its expression under polyhedrin (polh) promoter; however, this conclusion was not tested on different IMPs to generalize such a conclusion. In this study, the expression of eight different IMPs has been compared under vp39 and polh promoters of Autographa californica nucleopolyhedrovirus. Although different IMPs have shown different patterns of expression, the expression driven by vp39 promoter was found to be generally more efficient than the polh promoter. 相似文献
18.
Modeling of the structural features of integral-membrane proteins reverse-environment prediction of integral membrane protein structure (REPIMPS) 总被引:1,自引:0,他引:1
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Dastmalchi S Morris MB Church WB 《Protein science : a publication of the Protein Society》2001,10(8):1529-1538
The Profiles-3D application, an inverse-folding methodology appropriate for water-soluble proteins, has been modified to allow the determination of structural properties of integral-membrane proteins (IMPs) and for testing the validity of solved and model structures of IMPs. The modification, known as reverse-environment prediction of integral membrane protein structure (REPIMPS), takes into account the fact that exposed areas of side chains for many residues in IMPs are in contact with lipid and not the aqueous phase. This (1) allows lipid-exposed residues to be classified into the correct physicochemical environment class, (2) significantly improves compatibility scores for IMPs whose structures have been solved, and (3) reduces the possibility of rejecting a three-dimensional structure for an IMP because the presence of lipid was not included. Validation tests of REPIMPS showed that it (1) can locate the transmembrane domain of IMPs with single transmembrane helices more frequently than a range of other methodologies, (2) can rotationally orient transmembrane helices with respect to the lipid environment and surrounding helices in IMPs with multiple transmembrane helices, and (3) has the potential to accurately locate transmembrane domains in IMPs with multiple transmembrane helices. We conclude that correcting for the presence of the lipid environment surrounding the transmembrane segments of IMPs is an essential step for reasonable modeling and verification of the three-dimensional structures of these proteins. 相似文献
19.
Bensalem N Masscheleyn S Mozo J Vallée B Brouillard F Trudel S Ricquier D Edelman A Guerrera IC Miroux B 《Journal of proteome research》2007,6(4):1595-1602
Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents. 相似文献
20.
Lyukmanova EN Shenkarev ZO Khabibullina NF Kopeina GS Shulepko MA Paramonov AS Mineev KS Tikhonov RV Shingarova LN Petrovskaya LE Dolgikh DA Arseniev AS Kirpichnikov MP 《Biochimica et biophysica acta》2012,1818(3):349-358
Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. 相似文献