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 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.

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 The influence of the extracellular domains on the dynamic behavior of membrane proteins

2021 ◽  
Author(s):  
Linda Wedemann ◽  
Cenk Onur Gurdap ◽  
Taras Sych ◽  
Erdinc Sezgin
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Dynamics ◽  
Dynamic Behavior ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Cortical Actin ◽  
Extracellular Domains ◽  
The Impact

The dynamic behavior of the plasma membrane proteins mediates various cellular processes, such as cell-cell interactions, transmembrane transport and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interaction of the intracellular domain with cytosolic components such as cortical actin. However, the impact of the extracellular domains (ECDs) on the dynamics of membrane proteins is rather unexplored. Here, we investigate how the ECD size influences protein dynamics in lipid bilayer. We reconstitute ECDs of different molecular weights and heights in model membrane systems and analyze ECD-driven protein sorting in lipid domains as well as protein mobility. We observe that increasing the ECD size leads to a decrease in ordered domain partitioning as well as diffusivity. Our data suggests a critical role of the ECDs on membrane protein behavior in the plasma membrane and paves the way to a more complete understanding of membrane protein dynamics that includes interaction with the extracellular matrix and glycocalyx in health and disease.

Fluorescent potentiometric dyes for membrane-potential imaging

1994 ◽  
Vol 52 ◽  
pp. 166-167
Author(s):  
Leslie M. Loew
Keyword(s):  
Membrane Potential ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Optical Recording ◽  
Biological Processes ◽  
Major Focus ◽  
The Past ◽  
Excitable Systems ◽  
Major Application ◽  
The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

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 The Impact of Gelatin on the Pharmaceutical Characteristics of Fucoidan Microspheres with Posaconazole

Materials ◽  
2021 ◽  
Vol 14 (15) ◽  
pp. 4087
Author(s):  
Marta Szekalska ◽  
Aleksandra Citkowska ◽  
Magdalena Wróblewska ◽  
Katarzyna Winnicka
Keyword(s):  
Antifungal Activity ◽  
Drug Release ◽  
Spray Drying ◽  
Fungal Infections ◽  
Drug Loading ◽  
High Surface ◽  
Vaginal Fluid ◽  
Sustained Drug Release ◽  
Candida Spp ◽  
The Impact

Fungal infections and invasive mycoses, despite the continuous medicine progress, are an important globally therapeutic problem. Multicompartment dosage formulations (e.g., microparticles) ensure a short drug diffusion way and high surface area of drug release, which as a consequence can provide improvement of therapeutic efficiency compared to the traditional drug dosage forms. As fucoidan is promising component with wide biological activity per se, the aim of this study was to prepare fucospheres (fucoidan microparticles) and fucoidan/gelatin microparticles with posaconazole using the one-step spray-drying technique. Pharmaceutical properties of designed fucospheres and the impact of the gelatin addition on their characteristics were evaluated. An important stage of this research was in vitro evaluation of antifungal activity of developed microparticles using different Candida species. It was observed that gelatin presence in microparticles significantly improved swelling capacity and mucoadhesiveness, and provided a sustained POS release. Furthermore, it was shown that gelatin addition enhanced antifungal activity of microparticles against tested Candida spp. strains. Microparticles formulation GF6, prepared by the spray drying of 20% fucoidan, 5% gelatin and 10% Posaconazole, were characterized by optimal mucoadhesive properties, high drug loading and the most sustained drug release (after 8 h 65.34 ± 4.10% and 33.81 ± 5.58% of posaconazole was dissolved in simulated vaginal fluid pH 4.2 or 0.1 M HCl pH 1.2, respectively).

scholarly journals Brain drain and brain gain in Russia: Analyzing international migration of researchers by discipline using Scopus bibliometric data 1996–2020

2021 ◽  
Author(s):  
Alexander Subbotin ◽  
Samin Aref
Keyword(s):  
Brain Drain ◽  
Policy Development ◽  
Donor Country ◽  
International Mobility ◽  
Science System ◽  
Migration Rates ◽  
Net Migration ◽  
Novel Approach ◽  
Five Disciplines ◽  
The Impact

AbstractWe study international mobility in academia, with a focus on the migration of published researchers to and from Russia. Using an exhaustive set of over 2.4 million Scopus publications, we analyze all researchers who have published with a Russian affiliation address in Scopus-indexed sources in 1996–2020. The migration of researchers is observed through the changes in their affiliation addresses, which altered their mode countries of affiliation across different years. While only 5.2% of these researchers were internationally mobile, they accounted for a substantial proportion of citations. Our estimates of net migration rates indicate that while Russia was a donor country in the late 1990s and early 2000s, it has experienced a relatively balanced circulation of researchers in more recent years. These findings suggest that the current trends in scholarly migration in Russia could be better framed as brain circulation, rather than as brain drain. Overall, researchers emigrating from Russia outnumbered and outperformed researchers immigrating to Russia. Our analysis on the subject categories of publication venues shows that in the past 25 years, Russia has, overall, suffered a net loss in most disciplines, and most notably in the five disciplines of neuroscience, decision sciences, mathematics, biochemistry, and pharmacology. We demonstrate the robustness of our main findings under random exclusion of data and changes in numeric parameters. Our substantive results shed light on new aspects of international mobility in academia, and on the impact of this mobility on a national science system, which have direct implications for policy development. Methodologically, our novel approach to handling big data can be adopted as a framework of analysis for studying scholarly migration in other countries.

scholarly journals A process-based method for predicting lateral erosion rates

2021 ◽  
Author(s):  
Myron van Damme
Keyword(s):  
Soil Mechanics ◽  
High Surface ◽  
Shear Stresses ◽  
Empirical Equations ◽  
Predictive Capability ◽  
Surface Shear ◽  
Erosion Rates ◽  
Material Texture ◽  
Empirical Coefficients ◽  
The Impact

AbstractAn accurate means of predicting erosion rates is essential to improve the predictive capability of breach models. During breach growth, erosion rates are often determined with empirical equations. The predictive capability of empirical equations is governed by the range for which they have been validated and the accuracy with which empirical coefficients can be established. Most empirical equations thereby do not account for the impact of material texture, moisture content, and compaction energy on the erosion rates. The method presented in this paper acknowledges the impact of these parameters by accounting for the process of dilation during erosion. The paper shows how, given high surface shear stresses, the erosion rate can be quantified by applying the principles of soil mechanics. Key is thereby to identify that stress balance situation for which the dilatency induced inflow gives a maximum averaged shear resistance. The effectiveness of the model in predicting erosion rates is indicated by means of three validation test cases. A sensitivity analysis of the method is also provided to show that the predictions lie within the range of inaccuracy of the input parameters.

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