Name: site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels
Uploaded: 2016-07-04T15:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

Biz yazının kritik okuma ve yorumlar için Chakrapani laboratuar mevcut ve eski üyeleri için çok müteşekkiriz. Bu çalışma Ulusal Sağlık hibe Enstitüleri (1R01GM108921) ve Amerikan Kalp Derneği (NCRP Scientist Kalkınma Hibe 12SDG12070069) tarafından ve SC desteklenmiştir.

The authors have nothing to disclose.

EPR Spektroskopisi yakın bir doğal ortamında membran proteinleri yapısal değişimlere miktarının bir benzeri olmayan yapısal bir yaklaşım olduğu kanıtlanmıştır. Bu yaklaşım bize X-ışını kristalografisi ve Cryo-elektron mikroskobu yüksek çözünürlüklü yapılarda gizlenmiş protein dinamikleri moleküler detaya bir göz sağlar. Ancak, diğer sistemlere genel uygulanabilirliğini etkileyebilecek ve aynı zamanda akılda yol boyunca potansiyel deneysel barikatlar tutmak için bu yaklaşımın tek...

Son on yılda, pentameric ligand-kapılı iyon kanallarının yapısal anlayış (pLGIC) ailesinin bazı üyeleri yüksek çözünürlüklü yapıların yığınlarının nedeniyle, çarçabuk büyüdü. Alandaki güncel gelişmelere yol açan temel faktörler, yapı tayinleri yaklaşımları prokaryotik pLGIC kanallarının keşif, ökaryot membran proteini ifadesinde 1-3 büyük ilerlemeler, 4-6 ve muazzam atılımlar bulunmaktadır. 7 Bu yapılar net bir fikir birliği sağlamak pLGIC üç boyutlu mimari genel koruma. Ancak, geride iz gibi iki önemli alanlar bu kanal hazırlıkları fonksiyonel karakterizasyonu ve kanal fonksiyonu mekanik açıklaması vardır. Gating konformasyonel değişiklikler karmaşıktır ve kanalın uzunluğu boyunca 60 bir mesafede meydana gelmekte, bu geçişler yaygın olarak modüle edilirmembran lipidleri. Özel olarak, negatif lipidler, kolesterol ve fosfolipid pLGIC 8-11 fonksiyonunu modüle ettiği gösterilmiştir. Kanal fonksiyonunda bu lipit bileşenlerin kesin rolü bilinmese de, yolluk tam bir moleküler anlayış kendi doğal ortamında bu kanallar okuyan gerektirecektir. Site Yönetmen Spin Etiketleme (SDSL) ve Elektron Paramagnetik Rezonans (EPR) spektroskopisi sulandırılmış sistemlerde protein dinamikleri incelemek için seçim teknikleridir. (Floresans spektroskopisi olduğu gibi) ve bu şekilde doğal lipit koşulları içinde yeniden tam uzunluktaki konstruktlarının ölçümleri sağlar EPR spektroskopisi molekül büyüklüğü ile sınırlıdır (NMR olduğu gibi) ya da numune optik özellik değildir. teknik son derece hassas ve (pico-mol aralığında) nispeten düşük örnek gereksinimleri vardır. Bu iki yönü miligram üzerinde ifade etmek zordur büyük membran proteinleri eğitim tekniği uygundur yapmakmiktarları. Mevkiye-yönelik sıkma etiketi ile kombinasyon halinde EPR spektroskopi kullanılması Wayne Hubbell ve arkadaşları tarafından geliştirilen, ve protein tiplerine çalışmak için adapte edilmiştir. 12-24 EPR veriler ikincil yapılar araştırmak için kullanılmıştır, protein değişiklikleri konformasyon, zara sokma derinliği ve protein-protein / protein-ligand etkileşimleri. yöntem, alana-yönelik mutagenez ile ilgi pozisyonlarda sistein ikame içerir. Site-spesifik etiketleme sağlamak için, bir sistein az bir şablon oluşturmak için bir başka amino asit ile (ör., Serin) yerel sistein yerine gereklidir. bir disülfid bağı köprü ile proteine ​​bağlanır (1-oksil-2,2,5,5-tetrametil-Δ3-pirolin-3-metil) methanethiosulfonate: Bugüne kadar, en popüler spin etiket bir tiol özgü MTSL olup. Nedeniyle yüksek özgüllüğü, (triptofan biraz daha büyük) nispeten küçük boyutu ve flexi içinbağlayıcı bölgenin ortaya koymak bu spin etiket bile toprağa sistein ile mükemmel bir reaktiviteye sahip olduğu gösterilmiştir. Ayrıca, reaktiviteyi arttırmak için, bir proteinin İşaretleme reaksiyonu deterjan çözünmüş formda gerçekleştirilir. boyut dışlama kromatografisi ile fazla serbest yan etiketin ayrılmasından sonra, protein, lipozomlar veya tanımlanan lipid bileşimin iki tabakalı taklit eden sistemlere yeniden oluşturulur. Genel olarak, sistein mutagenez, protein birçok yerinde tolere edilir, ve spin sondası nispeten küçük ikincil ve üçüncül yapıları çok az pertürbasyon neden olur. modifikasyon vahşi tip fonksiyonları tutulmasını sağlamak için, etiketli ve yeniden kanal yama-kelepçe ölçümleriyle ele alınabilir. Etiketli fonksiyonlu protein daha sonra esas olarak üç ana bilgi tiplerini sağlayan spektroskopik ölçümler, tabi tutulmuştur: linesh göre 12,14,15,20,22,23,25-27 spin sondası dinamiklerimaymun analizi; paramanyetik gevşeme ajanlara prob erişilebilirlik; ve mesafe dağılımı. 27 EPR mesafeleri iki farklı yaklaşım ile ölçülür. İlk spin-etiketler arasında iki kutuplu ilişkilerden kaynaklanan spektral genişlemesi (8 - 20 Å mesafe aralığında) Sürekli Dalga (CW) tekniğine dayanmaktadır. Mesafeyi belirlemek için kullanılan 28,29 saniye darbeli bir-EPR olduğunu mesafe ölçümleri Çift Elektron Elektron Rezonans (geyik) ise 70 Å. 30-34 kadar uzatılabilir yöntem, spin-eko genlik salınımları mesafeleri ve mesafe dağılımlarını belirlemek için analiz edilir. İşte spin eko dipolar etkileşim frekansta modüle edilir. Birlikte, bu parametreler, protein topolojisi, ikincil yapısal elemanlar ve protein yapı değişiklikleri belirlemek için kullanılır.

<table><tbody><tr><td>Site-Directed Mutagenesis and Cys mutations</td><td></td><td></td><td></td></tr><tr><td>10x PfuUltra HF reaction buffer</td><td>Agilent Technologies</td><td>600380-52</td><td></td></tr><tr><td>dNTPS</td><td>New England BioLabs Inc&amp;lrm;</td><td>N0447L</td><td>10 mM each dNTP</td></tr><tr><td>pfu Ultra DNA polymerase</td><td>Agilent Technologies</td><td>600380-51</td><td>2.5 U/ul</td></tr><tr><td>DPNI</td><td>New England BioLabs Inc&amp;lrm;</td><td>R0176S</td><td>20,000 U/ml</td></tr><tr><td>XL10 GOLD</td><td>Agilent Technologies</td><td>200314</td><td></td></tr><tr><td>SOC media</td><td>New England BioLabs Inc&amp;lrm;</td><td>B9020S</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher Scientfic</td><td>BP905</td><td></td></tr><tr><td>LB media</td><td>Invitrogen</td><td>127957084</td><td></td></tr><tr><td>Miniprep kit</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>C43 competent cells</td><td>Lucigen</td><td>60446</td><td></td></tr><tr><td>Expression and Purification</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td>Fisher Scientfic</td><td>D16</td><td></td></tr><tr><td>Tryptone</td><td>Fisher Bioreagents</td><td>BP1421-500</td><td></td></tr><tr><td>Yeast extract</td><td>Amresco</td><td>J850</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Bioreagents</td><td>BP229</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0705</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0781</td><td></td></tr><tr><td>IPTG (isopropyl-thio-&amp;beta;-galactoside)</td><td>Gold Biotechnology</td><td>I2481C25</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma Life Science</td><td>T1503</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>DNase I</td><td>Sigma Life Science</td><td>DN25</td><td></td></tr><tr><td>PMSF</td><td>Amresco</td><td>M145</td><td></td></tr><tr><td>Leupeptine</td><td>Amresco</td><td>J580</td><td></td></tr><tr><td>Pepstatin</td><td>Amresco</td><td>J583</td><td></td></tr><tr><td>DDM (n-Docecyl-&amp;beta;-D-Maltopyranoside)</td><td>Anatrace</td><td>D310S</td><td></td></tr><tr><td>Amylose resin</td><td>New England BioLabs Inc&amp;lrm;</td><td>E8021L</td><td></td></tr><tr><td>TCEP</td><td>Amresco</td><td>K831</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientfic</td><td>BP118</td><td></td></tr><tr><td>Maltose</td><td>Acros Organics</td><td>329915000</td><td></td></tr><tr><td>Superdex 200 GL</td><td>GE Healthcare</td><td>17-5175-01</td><td></td></tr><tr><td>Empty polypropylene Chromatography column</td><td>BioRad</td><td>731-1550</td><td></td></tr><tr><td>Site-Directed Spin Labeling</td><td></td><td></td><td></td></tr><tr><td>MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate</td><td>Toronto Reaserch chemicals Inc</td><td>O873900</td><td></td></tr><tr><td>(1-acetoxy-2,2,5,5-tetramethyl-&amp;Delta;3-pyrroline-3-methyl) methanethiosulfonate</td><td>Toronto Reaserch chemicals Inc</td><td>A167900</td><td></td></tr><tr><td>DMSO</td><td>J.T. Baker</td><td>9224-01</td><td></td></tr><tr><td>Reconstitution</td><td></td><td></td><td></td></tr><tr><td>Asolectin lipid</td><td>Avanti polar lipids Inc</td><td>541602C</td><td></td></tr><tr><td>Biobeads (Polystyrine beads)</td><td>Bio Rad</td><td>152-3920</td><td></td></tr><tr><td>Methanol</td><td>Fisher chemicals</td><td>A413</td><td></td></tr><tr><td>FRET</td><td></td><td></td><td></td></tr><tr><td>Fluorescein-maleimide</td><td>ThermoFisher Scientific</td><td>F-150</td><td></td></tr><tr><td>Tetramethylrhodamine-maleimide</td><td>ThermoFisher Scientific</td><td>T-6027</td><td></td></tr><tr><td>POPC</td><td>Avanti polar lipids Inc</td><td>850457C</td><td></td></tr><tr><td>POPG</td><td>Avanti polar lipids Inc</td><td>840457C</td><td></td></tr><tr><td>&lt;em&gt;E.coli&lt;/em&gt; polar lipid extract</td><td>Avanti polar lipids Inc</td><td>100600C</td><td></td></tr><tr><td>HEPES</td><td>Sigma Life Science</td><td>H3375</td><td></td></tr><tr><td>EPR measurement</td><td></td><td></td><td></td></tr><tr><td>TPX plastic capillaries</td><td>Bruker</td><td>ER221</td><td></td></tr><tr><td>EDDA (Ethylenediamine-N, N&#039;-diacetic acid)</td><td>Aldrich</td><td>158186</td><td></td></tr><tr><td>Ni(OH)&lt;sub&gt;2&lt;/sub&gt;</td><td>Aldrich</td><td>283622</td><td></td></tr></tbody></table>

site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

Spin işaretli GLIC Mutantlarının Biyokimyasal Karakterizasyonu kültür, 12 mg / L - tipik olarak 10 ila GLIC-MBP füzyon proteinini doğuracak, yukarıda tarif edilen protokol izlenerek. Bu değer, özellikle protein içinde gömülü pozisyonlar için, farklı mutantların arasında değişse de, verim önemli ölçüde bozulabilir. Bu gibi durumlarda, kültür hacimleri ölçekleme gerektirebilir. HRV3C ...

Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Site Yönetmen Spin Etiketleme ve pentameric ligand-kapılı iyon Kanallarının EPR Spektroskopik Çalışmaları

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

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Research

JoVE Journal

Chemistry

10.6K Görüntüleme.  Case Western Reserve University. This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Video: Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

Biyomolekül Yapısının Belirlenmesi İçin Tek Molekül Seviyesinde Yüksek Hassasiyetteki FRET

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biyokimya

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

Bir iyon kanal protein biyo-katman Interferometry tahlil tarafından küçük molekülleri ile etkileşimi Kinetik yakalama

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Arıtma ve TRPV1 sulandırma spektroskopik analizi için

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lüminesans Rezonans Enerji memeli hücrelerinde ifade Membran Proteinleri Konformasyonel Değişiklikleri Eğitim transfer

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Biyomühendislik

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Sağlam, fonksiyonel membran proteinlerine bağlanan nükleotidin gerçek zamanlı olarak ölçülmesi

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

İçsel Olarak Düzensiz Proteinlerin Kendi Kendine İlişkilerini Tespit Etmek ve Karakterize Etmek için Paramanyetik Gevşeme Geliştirmesi

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

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

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

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

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

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

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 ...

Biyoloji

Makromoleküllerin CryoEM Haritalarında Küçük Moleküllü Ligandların Modellenmesi

Modeling Ligands into Maps Derived from Electron Cryomicroscopy

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological processes, and drug development. In recent years, cryogenic sample electron microscopy (cryoEM) has emerged as a powerful technique to determine the structures of macromolecules and to investigate the mode of ligand binding at near-atomic resolution. Identifying and modeling non-protein molecules in cryoEM maps is often challenging due to anisotropic resolution across the molecule of interest and inherent noise in the data. In this article, the readers are introduced to various software and methods currently used for ligand identification, model building, and refinement of atomic coordinates using selected macromolecules. One of the simplest ways to identify the presence of a ligand, as illustrated with the enolase enzyme, is to subtract the two maps obtained with and without the ligand. The extra density of the ligand is likely to stand out in the difference map even at a higher threshold. There are instances, as shown in the case of metabotropic Glutamate receptor mGlu5, when such simple difference maps cannot be generated. The recently introduced method of deriving the Fo-Fc omit map can serve as a tool for validating and demonstrating the presence of the ligand. Finally, using the well-studied &#946;-galactosidase as an example, the effect of resolution on modeling the ligands and solvent molecules in cryoEM maps is analyzed, and an outlook on how cryoEM can be used in drug discovery is presented.

Yazar Spotlight: Cryo-EM Haritalarında Ligandları Modelleyerek Hücresel Süreçleri Keşfetmek

This protocol introduces the tools available for modeling small-molecule ligands in cryoEM maps of macromolecules.

Ligandların Elektron Kriyomikroskopisinden Türetilen Haritalara Modellenmesi

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological ...

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