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.

scholarly journals Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Paul Curnow ◽  
Benjamin J. Hardy ◽  
Virginie Dufour ◽  
Christopher J. Arthur ◽  
Richard Stenner ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
De Novo ◽  
Zinc Protoporphyrin ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
Starting Point ◽  
Helix Packing ◽  
The Impact

Abstract Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

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.

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.

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 Context-specific action of macrolide antibiotics on the eukaryotic ribosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maxim S. Svetlov ◽  
Timm O. Koller ◽  
Sezen Meydan ◽  
Vaishnavi Shankar ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Amino Acid Sequences ◽  
Macrolide Antibiotics ◽  
Single Mutation ◽  
Sequence Motifs ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
Cellular Translation ◽  
Distinct Sequence ◽  
Exit Tunnel ◽  
Context Specific

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

scholarly journals Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Cheng-Wen He ◽  
Xue-Fei Cui ◽  
Shao-Jie Ma ◽  
Qin Xu ◽  
Yan-Peng Ran ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Molecular Mechanisms ◽  
Multivesicular Body ◽  
Degradation Process ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Membrane Structures ◽  
Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

scholarly journals A microfluidic strategy for the detection of membrane protein interactions

Lab on a Chip ◽  
2020 ◽  
Vol 20 (17) ◽  
pp. 3230-3238
Author(s):  
Yuewen Zhang ◽  
Therese W. Herling ◽  
Stefan Kreida ◽  
Quentin A. E. Peter ◽  
Tadas Kartanas ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Interactions ◽  
Native Solution ◽  
Exchange Of Information ◽  
Extracellular Environment

Membrane proteins are gatekeepers for exchange of information and matter between the intracellular and extracellular environment. This paper opens up a route to probe membrane protein interactions under native solution conditions using microfluidics.

Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

Sign in / Sign up

Export Citation Format

Share Document