scholarly journals Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yu-Hung Lin ◽  
Sung-Yao Lin ◽  
Guan-Syun Li ◽  
Shao-En Weng ◽  
Shu-Ling Tzeng ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Information ◽  
Solvent Accessibility ◽  
Band Shift ◽  
Fluorescent Band ◽  
Novel Approach ◽  
Target Membrane ◽  
Bile Acid Transporter ◽  
Membrane Spanning

Abstract The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis. Here, we report a novel approach, site-directed alkylation detected by in-gel fluorescence (SDAF), which monitors the fluorescent band shift caused by alkylation of the EGFP-fused target membrane protein bearing one single introduced cysteine. In-gel fluorescence provides a unique readout of target membrane proteins with EGFP fusion from non-purified samples, revealing a distinct 5 kDa shift on SDS-PAGE gel due to conjugation with mPEG-MAL-5K. Using the structurally characterised bile acid transporter ASBTNM as an example, we demonstrate that SDAF generates a topology map consistent with the crystal structure. The efficiency of mPEG-MAL-5K modification at each introduced cysteine can easily be quantified and analysed, providing a useful tool for probing the solvent accessibility at a specific position of the target membrane protein.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

scholarly journals Testing Parameters for Two-Dimensional Crystallization and Electron Crystallography on Eukaryotic Membrane Proteins with Liposomes as Controls

2008 ◽  
Vol 16 (4) ◽  
pp. 30-33
Author(s):  
Gengxiang Zhao ◽  
Vasantha Mutucumarana ◽  
Darrel W. Stafford ◽  
Yoshihide Kanaoka ◽  
K. Frank Austen ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Drug Targets ◽  
Structural Information ◽  
Protein Solubility ◽  
Three Dimensional ◽  
Phospholipid Bilayer ◽  
Structural Level ◽  
Electron Crystallography

Membrane proteins comprise the majority of known and potential drug targets, yet have been immensely difficult to analyze at the structural level due to their location in the membrane bilayer. Removal from the membrane necessitates replacement of the phospholipid bilayer by detergents in order to maintain protein solubility. However, the absence of lipids and the presence of detergents can render non-physiological conformational changes of the membrane protein (Tate, 2006). Electron crystallography is an important method for studying membrane proteins that usually takes advantage of reconstituting the protein in a phospholipid bilayer and removal of the detergent. Richard Henderson and Nigel Unwin used this technique to elucidate the three-dimensional (3D) arrangement of the transmembrane α-helices of bacteriorhodopsin, which was the first 3D structural information on a membrane protein (Henderson and Unwin, 1975).

scholarly journals Oxidative footprinting in the study of structure and function of membrane proteins: current state and perspectives

2015 ◽  
Vol 43 (5) ◽  
pp. 983-994 ◽  
Author(s):  
Vassiliy N. Bavro ◽  
Sayan Gupta ◽  
Corie Ralston
Keyword(s):  
Membrane Proteins ◽  
Structural Biology ◽  
Conformational Changes ◽  
Structural Information ◽  
Solvent Accessibility ◽  
Structure And Function ◽  
Specific Information ◽  
High Resolution Data ◽  
And Function ◽  
High Degree

Membrane proteins, such as receptors, transporters and ion channels, control the vast majority of cellular signalling and metabolite exchange processes and thus are becoming key pharmacological targets. Obtaining structural information by usage of traditional structural biology techniques is limited by the requirements for the protein samples to be highly pure and stable when handled in high concentrations and in non-native buffer systems, which is often difficult to achieve for membrane targets. Hence, there is a growing requirement for the use of hybrid, integrative approaches to study the dynamic and functional aspects of membrane proteins in physiologically relevant conditions. In recent years, significant progress has been made in the field of oxidative labelling techniques and in particular the X-ray radiolytic footprinting in combination with mass spectrometry (MS) (XF–MS), which provide residue-specific information on the solvent accessibility of proteins. In combination with both low- and high-resolution data from other structural biology approaches, it is capable of providing valuable insights into dynamics of membrane proteins, which have been difficult to obtain by other structural techniques, proving a highly complementary technique to address structure and function of membrane targets. XF–MS has demonstrated a unique capability for identification of structural waters and conformational changes in proteins at both a high degree of spatial and a high degree of temporal resolution. Here, we provide a perspective on the place of XF–MS among other structural biology methods and showcase some of the latest developments in its usage for studying water-mediated transmembrane (TM) signalling, ion transport and ligand-induced allosteric conformational changes in membrane proteins.

scholarly journals Substrate discrimination by size-exclusion in the intramembrane protease RseP

2014 ◽  
Vol 70 (a1) ◽  
pp. C1497-C1497
Author(s):  
Yohei Hizukuri ◽  
Takashi Oda ◽  
Sanae Tabata ◽  
Keiko Tamura-Kawakami ◽  
Rika Oi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Active Center ◽  
3D Structure ◽  
Size Exclusion ◽  
Sequence Motif ◽  
Pdz Domains ◽  
Specific Sequence ◽  
X Ray Crystallography ◽  
Target Membrane ◽  
Membrane Spanning

Regulated intramembrane proteolysis (RIP), wherein a target membrane protein is specifically cleaved within the transmembrane region, is now accepted as a form of cellular signaling. As a result of proteolysis, a soluble portion of the target membrane protein is liberated to act as a signaling molecule. RIP is catalyzed by intramembrane-cleaving proteases, which are now classified into site-2 protease, rhomboid and γ-secretase/SPP families based on the mechanism of catalysis. E. coli possesses a site-2 protease homolog RseP, which is implicated in the extracytoplasmic stress response. RseP cleaves a membrane-spanning anti-σE protein RseA to release σE from the membrane, where truncation of the C-terminal periplasmic part of RseA by a membrane-anchored protease DegS triggers the action of RseP. Hence, there must be some mechanism by which RseP senses the DegS-cleavage of RseA. RseP possesses two tandemly-arranged PDZ domains (PDZ tandem) in the periplasmic region, which have been suggested to be involved in the regulation of cleavage. Although PDZ domains generally recognize the C-terminal sequence of a ligand, most of the previous works suggested that the RseP PDZ domains are involved in the suppression of the intramembrane cleavage of RseA. In this study, we determined the 3D structure of the PDZ tandem by X-ray crystallography and SAXS and showed that the two PDZ domains are arranged in an overall "clam-like" configuration to constitute a "pocket-like" structure. Sequence analysis suggested that the PDZ tandem would lie just above the active center sequestrated within the membrane. Furthermore, chemical modification demonstrated that the interior of the pocket is inaccessible to a bulky reagent in the full-length RseP. Taken together, we have made a proposal that RseP accommodates the truncated RseA into the active center by a steric size-exclusion mechanism through the PDZ tandem, rather than by recognition of a specific sequence/motif of RseA.

scholarly journals Structure Topology Prediction of Discriminative Sequence Motifs in Membrane Proteins with Domains of Unknown Functions

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Steffen Grunert ◽  
Florian Heinke ◽  
Dirk Labudde
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
In Silico Analysis ◽  
Sequence Motifs ◽  
Protein Families ◽  
Structure Topology ◽  
General Importance ◽  
Novel Approach ◽  
Distinct Sequence ◽  
High Prediction

Motivation. Membrane proteins play essential roles in cellular processes of organisms. Photosynthesis, transport of ions and small molecules, signal transduction, and light harvesting are examples of processes which are realised by membrane proteins and contribute to a cell's specificity and functionality. The analysis of membrane proteins has shown to be an important part in the understanding of complex biological processes. Genome-wide investigations of membrane proteins have revealed a large number of short, distinct sequence motifs. Results. The in silico analysis of 32 membrane protein families with domains of unknown functions discussed in this study led to a novel approach which describes the separation of motifs by residue-specific distributions. Based on these distributions, the topology structure of the majority of motifs in hypothesised membrane proteins with unknown topology can be predicted. Conclusion. We hypothesise that short sequence motifs can be separated into structure-forming motifs on the one hand, as such motifs show high prediction accuracy in all investigated protein families. This points to their general importance in α-helical membrane protein structure formation and interaction mediation. On the other hand, motifs which show high prediction accuracies only in certain families can be classified as functionally important and relevant for family-specific functional characteristics.

Bioenergetics at the gold surface: SEIRAS probes photosynthetic and respiratory reactions at the monolayer level

2008 ◽  
Vol 36 (5) ◽  
pp. 986-991 ◽  
Author(s):  
Kenichi Ataka ◽  
Joachim Heberle
Keyword(s):  
Membrane Potential ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
High Surface ◽  
Electrical Potential ◽  
Adsorbed Molecule ◽  
Gold Surface ◽  
Ir Absorption ◽  
Novel Approach ◽  
The Impact

The present study surveys a novel approach to studies of membrane proteins whose catalytic action is driven by the redox potential or by the membrane potential. We introduce SEIRAS (surface-enhanced IR absorption spectroscopy) to probe a monolayer of membrane protein adhered to the surface of a gold electrode. SEIRAS renders high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude. It is demonstrated that reaction-induced spectroscopy is applicable by recording IR differences of cytochrome c after stimulation by the electrical potential. The impact of the membrane potential on the function of a membrane protein is demonstrated by performing light-induced difference spectroscopy on a microbial rhodopsin (sensory rhodopsin II) under voltage-clamp conditions. The methodology presented opens new avenues to study the mechanism of electron-triggered and voltage-gated proteins at the level of single bonds. As many of these catalytic reactions are of vectorial nature, control on the orientation of the membrane protein is mandatory. Approaches are presented on how to specifically adhere photosynthetic and respiratory proteins to the electrode surface and reconstitute these membrane proteins in the lipid bilayer. Functionality of such biomimetic systems is assessed in situ by spectro-electrochemical methods.

scholarly journals Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

2000 ◽  
Vol 64 (1) ◽  
pp. 13-33 ◽  
Author(s):  
Marleen van Geest ◽  
Juke S. Lolkema
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Information ◽  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Membrane Topology ◽  
Protein Insertion ◽  
Membrane Protein Topology ◽  
Membrane Protein Insertion ◽  
Nascent Chain

SUMMARY Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

Simulation studies of the interactions between membrane proteins and detergents

2005 ◽  
Vol 33 (5) ◽  
pp. 910-912 ◽  
Author(s):  
P.J. Bond ◽  
J. Cuthbertson ◽  
M.S.P. Sansom
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Self Assembly ◽  
Kinetic Mechanism ◽  
Coarse Grained ◽  
Simulation Studies ◽  
High Resolution Data ◽  
Biologically Relevant ◽  
Structure And Dynamics ◽  
Detergent Interactions

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein–detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein–detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both α-helical and β-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein–detergent assemblies.

scholarly journals Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis

2021 ◽  
Vol 22 (7) ◽  
pp. 3793
Author(s):  
Sophie Blinker ◽  
Jocelyne Vreede ◽  
Peter Setlow ◽  
Stanley Brul
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Protein ◽  
Spore Germination ◽  
Structural Information ◽  
Transmembrane Protein ◽  
Homology Modelling ◽  
Water Channel ◽  
Md Simulations ◽  
Integral Membrane Protein ◽  
Starting Point

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.

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