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	<li>Cole, K. S., Moore, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+Ion+Current+in+the+Squid+Giant+Axon:+Dynamic+Characteristic.">Potassium Ion Current in the Squid Giant Axon: Dynamic Characteristic.</a> Biophys. J. 1, 1-14 (1960).</li><li>Taglialatela, M., Toro, L., Stefani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+voltage+clamp+to+record+small,+fast+currents+from+ion+channels+expressed+in+Xenopus+oocytes.">Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.</a> Biophys. J. 61, 78-82 (1992).</li><li>Dempski, R. E., Friedrich, T., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+subunit+of+the+Na+/K+-ATPase+follows+the+conformational+state+of+the+holoenzyme.">The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme.</a> J. Gen. Physiol. 125, 505-520 (2005).</li><li>Miller, A. J., Zhou, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+oocytes+as+an+expression+system+for+plant+transporters.">Xenopus oocytes as an expression system for plant transporters.</a> BBA - Biomembranes. 1465, 343-358 (2000).</li><li>Cha, A., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Voltage-Dependent+Conformational+Changes+in+the+Shaker+K++Channel+with+Fluorescence.">Characterizing Voltage-Dependent Conformational Changes in the Shaker K+ Channel with Fluorescence.</a> Neuron. 19, 1127-1140 (1997).</li><li>Mannuzzu, L. M., Moronne, M. M., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Physical+Measure+of+Conformational+Rearrangement+Underlying+Potassium+Channel+Gating.">Direct Physical Measure of Conformational Rearrangement Underlying Potassium Channel Gating.</a> Science. 271, 213-216 (1996).</li><li>Koch, H. P., Larsson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+molecular+motions+accomplish+glutamate+uptake+in+human+glutamate+transporters.">Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters.</a> J. Neurosci. 25, 1730-1736 (2005).</li><li>Dempski, R. E., Hartung, K., Friedrich, T., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorometric+Measurements+of+Intermolecular+Distances+between+the+&#945;-+and+&#946;-Subunits+of+the+Na+/K+-ATPase.">Fluorometric Measurements of Intermolecular Distances between the &#945;- and &#946;-Subunits of the Na+/K+-ATPase.</a> J. Biol. Chem. 281, 36338-36338 (2006).</li><li>Geys, S. A., Bamberg, E., Dempski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-Dependent+Effects+on+the+Conformational+Equilibrium+of+the+Na+,K+-ATPase+As+Monitored+by+Voltage+Clamp+Fluorometry.">Ligand-Dependent Effects on the Conformational Equilibrium of the Na+,K+-ATPase As Monitored by Voltage Clamp Fluorometry.</a> Biophys. J. 96, 4561-4561 (2009).</li><li>Geibel, S., Kaplan, J. H., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Dynamics+of+the+Na+/K+-ATPase+Probed+by+Voltage+Clamp+Fluorometry.">Conformational Dynamics of the Na+/K+-ATPase Probed by Voltage Clamp Fluorometry.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 964-969 (2003).</li><li>Lorenz, L., Pusch, M., Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteromultimeric+CLC+chloride+channels+with+novel+properties.">Heteromultimeric CLC chloride channels with novel properties.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 13362-13366 (1996).</li><li>Mruk, K., Kobertz, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+Novel+Activator+of+KCNQ1-KCNE1+K++Channel+Complexes.">Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes.</a> Biophys. J. 177a, (2009).</li><li>Kyte, J., Doolittle, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+diplaying+the+hydropathic+character+of+a+protein.">A method for diplaying the hydropathic character of a protein.</a> J. Mol. Biol. 157, 105-132 (1982).</li><li>Rakowski, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+movement+by+the+Na/K+pump+in+Xenopus+oocytes.">Charge movement by the Na/K pump in Xenopus oocytes.</a> J. Gen. Physiol. 101, 117-144 (1993).</li><li>Tsunoda, S. P., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glu+87+of+Channelrhodopsin-1+Causes+pH-dependent+Color+Tuning+and+Fast+Photocurrent+Inactivation.">Glu 87 of Channelrhodopsin-1 Causes pH-dependent Color Tuning and Fast Photocurrent Inactivation.</a> Photochem. Photobiol. 85, 564-569 (2009).</li><li>Cha, A., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+implications+of+fluorescence+quenching+in+the+Shaker+K++channel.">Structural implications of fluorescence quenching in the Shaker K+ channel.</a> J. Gen. Physiol. 112, 391-391 (1998).</li><li>F&#246;rster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ann Physik. 2, 55-75 (1948).</li><li>Dong, X., Stothard, P., Forsythe, I. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PlasMapper:+a+web+server+for+drawing+and+auto-annotating+plasmid+maps.">PlasMapper: a web server for drawing and auto-annotating plasmid maps.</a> Nucleic Acids Res. 32, W660-W664 (2004).</li></ol>

Biz tarif deneysel yaklaşım siteye özgü fluorofor etiketleme ve iki elektrot voltajı, membran proteinlerin yapı ve fonksiyon arasındaki ilişkiyi araştırmak için kelepçe birleştirir. Bu teknik, iyon taşıma sırasında membran proteinlerinin konformasyonel dinamikleri zaman çözüme bilgiler elde etmek için kullanılabilir. Ayrıca, bu yaklaşım, iyon pompaları, iyon kanalları ve taşıyıcıları gibi çeşitli proteinlerin ile çalışmak için uygun olabilir. Belirli bir kalıntı konformasyonel dinamiklerini araştırıyor ek olarak, bir holoenzyme içinde mesafe sınırlamaları belirlemek için floresan rezonans enerji transferi için de kullanmak mümkündür. Kalıntılarının yanı olarak ölçüm altbirimden arasındaki göreceli hareketi arasındaki uzaklıkları belirlenmesi yolluk mekanizmaları ile ilgili anahtar sorular çözmeye yardımcı olabilir.

<table border="1"><tbody><tr><th> Reaktif / Ekipman </th><th> Şirket </th><th> Reaktif / Ekipman </th><th> Şirket </th></tr><tr><td> 100W Tungsten Işık Kaynağı </td><td> Carl Zeiss Microimaging </td><td> floresein-5-maleimide </td><td> Invitrogen </td></tr><tr><td> 1B150F-4 </td><td> Dünya Hassas Aletler </td><td> Yüksek Saf-PCR Ekstraksiyon Kiti </td><td> Roche </td></tr><tr><td> 3,0-4,0 Ethicon Vicryl </td><td> Johnson &amp; Johnson </td><td> mMessage mMachine Kit </td><td> Ambion </td></tr><tr><td> 475AF40 eksitasyon filtresi </td><td> Omega Optik </td><td> MS-222 </td><td> Sigma-Aldrich </td></tr><tr><td> 505DRLP dikroik aynalar </td><td> Omega Optik </td><td> Nucleospin Plazmid seti </td><td> MACHEREY-Nagel </td></tr><tr><td> MACHEREY-Nagel </td><td> Omega Optik </td><td> Nanodrop 2000c Sprectrophotometer </td><td> Thermo Scientific </td></tr><tr><td> 535DF50 eksitasyon filtresi </td><td> Omega Optik </td><td> PC-10 Mikropipet Çektirme </td><td> Narishige </td></tr><tr><td> 560DRLP dikroik aynalar </td><td> Omega Optik </td><td> 10 Yazılım pCLAMP </td><td> Akson Aletleri </td></tr><tr><td> 565ALP emisyon filtresi </td><td> Omega Optik </td><td> PfuTurbo DNA Polimeraz </td><td> Strategene </td></tr><tr><td> 565EFLP emisyon filtresi </td><td> Omega Optik </td><td> PIN-022A Photodiode </td><td> Birleşik Dedektör Teknolojileri </td></tr><tr><td> 570DRLP dikroik aynalar </td><td> Omega Optik </td><td> Polarize Filtreler </td><td> Linos Phtonics Inc. </td></tr><tr><td> Linos Phtonics Inc. </td><td> Omega Optik </td><td> QuickChange Site Yönetmen Mutagenisis Seti </td><td> Stratagene </td></tr><tr><td> Axio Examiner Floresan Mikroskobu </td><td> Carl Zeiss MicroImaging GMBH </td><td> RC-10 Floresans Odası </td><td> Warner Aletleri </td></tr><tr><td> Warner Aletleri </td><td> Yaşam Teknolojileri </td><td> tetramethylrhodamine-6-maleimide </td><td> Invitrogen </td></tr><tr><td> Digidata 1440A veri toplama sistemi </td><td> Akson Aletleri </td><td> TOP10 Electrocompetent hücre </td><td> Invitrogen </td></tr><tr><td> DPN I </td><td> New England Biolabs </td><td> Turbo Tec-05X amplifikatör </td><td> NPI </td></tr></tbody></table>

examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling

İki elektrot voltajı kelepçe elektrofizyoloji (TEVC), iyon kanalları 2, iyon pompaları 3, ve nakliyecileri 4 de dahil olmak üzere geniş bir yelpazede zarı proteinleri iyon transport1 mekanizmasını araştırmak için güçlü bir araçtır. Son gelişmeler, aynı anda tek hücre yüzeyinde bu proteinlerin belirli artıkları ve fonksiyon konformasyonel dinamiklerini incelemek için TEVC yanında siteye özgü fluorofor etiketleme kombine var. Biz membran proteinlerin aynı anda, voltaj kelepçe Florimetri kullanılarak floresans ve akım değişiklikleri izleme konformasyonel dinamiklerini incelemek için bir yöntem anlatacağım. Bu yaklaşım, site özellikle aşağıdaki sistein değiştirme ve site yönetmenliğini fluorofor etiketleme 5,6 membran proteinlerinin moleküler hareket incelemek için kullanılabilir. Ayrıca, bu yöntem, özel kalıntıları arasındaki mesafe 7,8 kısıtlamaları belirlemek için bir yaklaşım sağlar . Bu ilgi iki mutasyona uğramış sistein kalıntıları için, verici ve alıcı fluorophores seçici ekleyerek elde edilir. Kısacası, bu deneyler Xenopus Leavis oosit yüzeyinde istenen proteinin fonksiyonel ifade yapılmaktadır. Bu oositlerin geniş yüzey alanı facile fonksiyonel ölçümleri ve sağlam bir floresan sinyal 5 sağlar. Bu kolayca membran proteinlerinin 4 mekanizma hakkında daha fazla bilgi sağlayabilir, pH, ligand katyon / anyon gibi ekstrasellüler koşulları değiştirmek için de mümkündür . Son olarak, son gelişmeler, aynı zamanda ikinci bir protein 9 ile birlikte ifade seçin iç iyonlarının manipülasyon sağlamıştır. Bizim protokol birden fazla parça açıklanmıştır. İlk olarak, fluorofor etiketleme transmembran ve ekstrasellüler etki alanı arayüzünde bulunan kalıntılarının sistein tarama mutagenez ilerledi. Daha sonraki deneyler, protein konformasyonel değişiklik üzerine floresan (&lt;% 5) 3 büyük değişiklikler göstermektedir artıkları belirlemek için tasarlanmıştır. İkincisi, floresan yoğunluğu bu değişiklikler için 10 protein işlevine konformasyonel dinamikleri ilişkili membran proteini kinetik parametreleri karşılaştırıldı. Bu hedef proteinin moleküler hareket sıkı bir biyofiziksel analiz sağlar. Son olarak, iki artıkları holoenzyme donör photodestruction yöntemler kullanarak mesafe kısıtlamaları belirlemek amacıyla bir donör ve alıcı fluorofor ile etiketlenmiş olabilir. Verici ve alıcı fluorofor ile etiketleme aşağıdaki protein alt birimlerinin göreli hareketlerini izlemek için de mümkündür.

Watch this Scientific Journal Video about Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling at JoVE.com

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Biz tek hücreleri üzerinde floresan kullanarak yapı değişiklikleri siteye özgü analizi ile birlikte membran proteinlerinin iyon taşıma kinetiği ölçen bir yöntem anlatacağım. Bu teknik, iyon kanalları ve iyon pompaları uyarlanabilir ve protein altbirimden arasındaki mesafe sınırlamaları belirlemek için kullanılabilir.

Membran Proteinleri Konformasyonel Dynamics incelenmesi In situ Site Floresan Etiketleme

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

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Research

JoVE Journal

Biology

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

14.6K Görüntüleme.  Worcester Polytechnic Institute. Biz tek hücreleri üzerinde floresan kullanarak yapı değişiklikleri siteye özgü analizi ile birlikte membran proteinlerinin iyon taşıma kinetiği ölçen bir yöntem anlatacağım. Bu teknik, iyon kanalları ve iyon pompaları uyarlanabilir ve protein altbirimden arasındaki mesafe sınırlamaları belirlemek için kullanılabilir.

Video: Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein etkileşimleri çalışmak için aracı olarak floresan Anizotropi

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Biyokimya

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Membran Protein katlanması, Termodinamik Floresans Spektroskopisi ile ölçülür.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Biyomühendislik

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Yöntem gözünüzde canlandırın ve Transmisyon Elektron Mikroskobu Membran Etkileşen Proteinler Analiz için

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Ticari Mikroskop Canlı Hücre Zarları Moleküler Difüzyon Kanunu Hızlı Floresans Görüntüleme Gönderen

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Kimya

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

Tek Moleküllü FRET Kullanarak Membran Reseptörlerinin Konformasyonel Dinamiğinin Görselleştirilmesi

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

<meta charset="utf8"></head><body>GTPase Atlastin'in Farklı Nükleotid Yükleme Durumlarında İzlenmesi

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule F&#246;rster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

<meta charset="utf8"></head><body>Dinamin Benzeri GTPaz Atlastininin Konformasyonel Dinamiklerini Gözlemlemek için Tek Moleküllü FRET Görüntüleme

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

Dinamin Benzeri GTPaz Atlastininin Konformasyonel Dinamiklerini Gözlemlemek için Tek Moleküllü FRET Görüntüleme

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

Floresan Mikroskopi ile Kesme Akışı Altında Tek Moleküllü Konformasyonel Değişikliklerin Karakterizasyonu

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

Membran Proteinleri Konformasyonel Dynamics incelenmesi In situ Site Floresan Etiketleme

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Dinamin Benzeri GTPaz Atlastininin Konformasyonel Dinamiklerini Gözlemlemek için Tek Moleküllü FRET Görüntüleme

Dinamin Benzeri GTPaz Atlastininin Konformasyonel Dinamiklerini Gözlemlemek için Tek Moleküllü FRET Görüntüleme

Floresan Mikroskopi ile Kesme Akışı Altında Tek Moleküllü Konformasyonel Değişikliklerin Karakterizasyonu