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

우리는 원고의 중요한 읽기와 의견의 차크라 실험실의 현재와 전 회원들에게 매우 감사하고 있습니다. 이 작품은 보건 부여의 국립 연구소 (1R01GM108921)와 미국 심장 협회 (NCRP 과학자 개발 그랜트 12SDG12070069)에 의해 SC에 지원되었다.

The authors have nothing to disclose.

EPR 스펙트럼을 거의 네이티브 환경에서 막 단백질의 구조적 변화를 정량화에서 타의 추종을 불허하는 구조적 접근 것으로 입증되었습니다. 이러한 접근 방식은 우리에게 X 선 결정학 및 곳을 알아내는 - 전자 현미경에서 고해상도 구조에 가려하는 단백질 역학의 분자 세부 사항에 엿봄을 할 수 있습니다. 그러나, 다른 시스템으로 일반적인 적용에 영향을 미칠 수 있으며 마음에 길을 따라 잠재적 ...

지난 10 년간, 펜타 머 리간드 관문 이온 채널의 구조 이해 (pLGIC)는 여러 가족 구성원의 고해상도 구조 군중에 의해, 비약적으로 성장했다. 분야에서 현재 발전을 주도하는 중요한 요소는 구조 결정 방법의 원핵 pLGIC 채널 발견, 진핵 생물의 세포막 단백질 발현 1-3 주요 진행됨 4-6 엄청난 돌파구를 포함한다. (7) 이러한 구조가 명확한 합의 제공 pLGIC의 3 차원 구조의 전체적인 보존. 그러나 뒤에 흔적 것 두 가지 주요 영역이 채널 준비의 기능적 특성 및 채널 기능의 기계 론적 설명이다. 게이팅 구조적 변화는 복잡하고, 채널의 길이를 따라 60 Å 거리에 걸쳐 발생하고 이러한 전환을 광범위에 의해 조절되는막 지질. 특히, 음의 지질, 콜레스테롤 및 인지질 pLGIC 8-11의 기능을 조절하는 것으로 나타났다. 채널 기능에서 이러한 지질 성분의 정확한 역할을 알 수없는 남아 있지만, 게이트의 완전한 분자 이해는 그 나라의 환경에서 이러한 채널을 연구 필요합니다. 사이트 감독 스핀 라벨 (SDSL) 및 전자 스핀 공명 (EPR) 분광법은 재구성 시스템에서 단백질 역학 연구를위한 선택의 기술이다. (형광 스펙트럼은 그대로), 이로써 천연 지질 조건 재구성 전장 구조체의 측정이 가능 EPR 분광법은 분자 크기에 의해 제한 (NMR처럼) 또는 시료의 광학 특성 아니다. 이 기술은 매우 민감하고 (피코 몰 범위에서) 상대적으로 낮은 샘플 요구 사항이 있습니다. 이 두 측면은 밀리그램 이상에서 표현하기 어려운 큰 막 단백질 연구에 대한 기술이 적합하다수량. 부위 - 스핀 표지와 함께 EPR 분광법의 사용 웨인 Hubbell의 연구진에 의해 개발되었고, 단백질 종류의 범위를 학습하도록하고있다. 12-24 EPR 데이터 이차 구조를 조사하기 위해 사용 된, 단백질의 변화 형태, 막 - 삽입 깊이 및 단백질 - 단백질 / 단백질 - 리간드 상호 작용. 상기 방법은 부위 - 특이 적 돌연변이 유발에 의해 주목 위치의 시스테인의 치환을 포함한다. 부위 - 특이 적 표지를 보장하기 위해, 시스테인이없는 템플릿을 생성하기 위해 다른 아미노산과 (예., 세린) 천연 시스테인을 대체 할 필요가있다. 이황화 결합 다리를 통해 부착 단백질 (1- 옥실-2,2,5,5- 테트라 메틸 - 피 롤린 Δ3 -3- 메틸) methanethiosulfonate : 지금까지, 가장 인기 스핀 라벨 티올 특정 MTSL이다. 그것의 높은 특이성, (트립토판보다 약간 큰) 비교적 작은 크기, 플렉시에링커 영역의 부 합성,이 스핀 라벨에도 매립 시스테인 우수한 반응성을 가지는 것으로 밝혀졌다. 또한, 반응성을 극대화하기 위해, 단백질의 표지 반응은 세제 - 가용화 된 형태로 수행된다. 크기 배제 크로마토 그래피에 의한 과잉 자유 스핀 라벨 분리 후, 단백질 또는 리포좀 정의 지질 조성물의 이중층 흉내 낸 시스템으로 재구성된다. 일반적으로, 시스테인 돌연변이가 아니라 단백질의 대부분의 부분에서 허용되고, 스핀 프로브의 상대적으로 작은 크기의 2 차 및 3 차 구조로 최소한의 혼란을 일으키는. 수정이 야생형 기능을 유지하도록하려면 표지 및 재구성 된 채널은 패치 클램프 측정에 의해 공부하실 수 있습니다. 표지 된 기능성 단백질은 다음 본질적으로 정보의 세 가지 주요 유형을 제공 분광 측정을 실시 : linesh에 의해 12,14,15,20,22,23,25-27 스핀 프로브 역학을원숭이 분석; 상자성 이완 에이전트 프로브의 접근성; 거리 분포. 27 EPR 거리는 두 개의 서로 다른 접근 방법에 의해 측정된다. 첫번째는 스핀 라벨 간의 극성 상호 작용에서 발생하는 스펙트럼의 폭이 넓어합니다 (8 - 20 Å 거리 범위) 연속파 (CW) 기술에 기초한다. 거리를 결정하는 데 사용되는 28, 29 번째 펄스-EPR 인 거리 측정 이중 전자 전자 공명 (DEER) 70 Å. 30-34까지 연장 될 수있어서, 상기 스핀 에코 진폭의 진동이 거리와의 거리 분포를 결정하기 위해 분석된다. 여기 스핀 에코는 쌍극 상호 작용의 주파수로 변조된다. 함께, 이러한 파라미터는 단백질 토폴로지 이차 구조 요소 및 단백질 구조적 변화를 결정하기 위해 사용된다.

<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.

스핀 표지 GLIC 변이체의 생화학 적 특성 배양 12 ㎎ / ℓ - 일반적으로 10 내지 GLIC-MBP 융합 단백질을 수득 할 상기 프로토콜에 따라. 이 값은 특히 단백질 내에 매립 위치에 대한 다른 돌연변이마다 다를 수 있지만, 수율이 크게 저하 될 수있다. 이 경우, 문화 볼륨 업 스케일링이 필요할 수 있습니다. HRV3C 프로테아제...

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.

사이트 감독 스핀 라벨 및 펜타 머 리간드 - 게이 티드 이온 채널의 EPR 분광 연구

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

site-directed-spin-labeling-epr-spectroscopic-studies-pentameric

Research

JoVE Journal

Chemistry

10.6K 조회수.  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.

비디오: 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.

생체 분자 구조 결정을위한 단일 분자 수준의 고정밀 FRET

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

연구

생화학

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.

바이오-레이어 간섭계 분석 결과 의해 작은 분자와 이온 채널 단백질의 상호 작용 활동을 캡처

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.

정화 및 분 광 분석에 대 한 TRPV1의 재구성

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.

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

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

생체공학

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.

온전한 기능성 막 단백질에 대한 뉴클레오티드 결합을 실시간으로 측정

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.

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins(본질적으로 무질서한 단백질의 자기 연관성을 검출하고 특성화하기 위한 상자성 이완 향상)

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 ...

화학

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.

막 단백질의 Conformational 역학 검사 현장에서 사이트 이동 형광 라벨링과

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

생물학

저분자 리간드(Small Molecule Ligands)의 CryoEM 맵 모델링

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.

저자 스포트라이트: Cryo-EM 맵에서 리간드를 모델링하여 세포 프로세스 탐색

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

Modeling Ligands into Maps Derived from Electron Cryomicroscopy(전자 극저온 현미경에서 파생된 맵으로 리간드 모델링)

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

사이트 감독 스핀 라벨 및 펜타 머 리간드 - 게이 티드 이온 채널의 EPR 분광 연구

요약

더 많은 비디오 탐색

이 비디오의 챕터

생체 분자 구조 결정을위한 단일 분자 수준의 고정밀 FRET

바이오-레이어 간섭계 분석 결과 의해 작은 분자와 이온 채널 단백질의 상호 작용 활동을 캡처

정화 및 분 광 분석에 대 한 TRPV1의 재구성

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

온전한 기능성 막 단백질에 대한 뉴클레오티드 결합을 실시간으로 측정

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins(본질적으로 무질서한 단백질의 자기 연관성을 검출하고 특성화하기 위한 상자성 이완 향상)

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

막 단백질의 Conformational 역학 검사 현장에서 사이트 이동 형광 라벨링과

Modeling Ligands into Maps Derived from Electron Cryomicroscopy(전자 극저온 현미경에서 파생된 맵으로 리간드 모델링)

Modeling Ligands into Maps Derived from Electron Cryomicroscopy(전자 극저온 현미경에서 파생된 맵으로 리간드 모델링)