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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8206;</td>
			<td>N0447L</td>
			<td>10mM 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&#8206;</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&#8206;</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>K2HPO4</td>
			<td>Amresco</td>
			<td>0705</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Amresco</td>
			<td>0781</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (isopropyl-thio-&#946;-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-&#946;-D-Maltopyranoside)</td>
			<td>Anatrace</td>
			<td>D310S</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England BioLabs Inc&#8206;</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 200GL</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-&#916;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>E.Coli 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&#39;-diacetic acid)</td>
			<td>Aldrich</td>
			<td>158186</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni(OH)2</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

Universidad San Sebastian

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

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

작은 올리고머 형성 막 단백질의 침전 평형 : 펜타 머 안정성에 히스티딘 양성자의 효과

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

연구

화학

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

토양 칼럼을 통해 표면 개질 된 카본 나노 튜브의 운송

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

기능성 잉크에 분산제 / 입자 결합 메커니즘의 반사율 적외선 분광 식별 확산

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

사용 하 여 순환 Voltammetry, UV-Vi-NIR, EPR Spectroelectrochemistry 유기 화합물 분석

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the methodology for non-substituted pyridines in 1957 in a 12 h process, but no X-ray suitable crystals were obtained. The substituted ring used in the methodology presented here clearly influenced the addition of water molecules into the asymmetric unit, which confers a different nucleophilic strength in 1. The X-ray suitable crystal compound 1 was possible due to the stabilization of the negative charge in the oxygen by the presence of two water molecules where the hydrogen atoms donate positive charge into the ring; such water molecules serve well to construct a supramolecular interaction. The hydrated molecules may be possible for the alkaline system that is reached by adjusting the pH to 10. Importantly, the double methyl substituted ring and a reaction time of 5 h, makes it a more versatile method and with wider chemical applications for future ring insertions.

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

Herein, we report the synthesis and crystallization of 3,5-lutidine N-oxide dehydrate by a simple protocol that differs from the classical synthesis of pyridine N-oxide. This protocol utilizes different starting material and involves less reaction time to yield a new solvated supramolecular structure, which crystallizes under slow evaporation.

종합, 결정 화, 그리고 3, 5-Lutidine N의 분 광 특성-산화물 탈수

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Azaheterocycles의 합성에 대 한 안정적인 Bicyclic Aziridinium 이온과 그들의 반지 개통 준비

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

막 거 대 한 소포 지역화 된 칼슘 이온 기울기에 대 한 응답에서의 개장

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

합성 및 Bioconjugation의 Thiol 반응 시 약 사이트에 선택적으로의 창조에 대 한 수정 Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

금속 결합 올리고펩타이드의 금속 이온 인식 및 Redox 활성의 구조 및 메커니즘을 결정하기 위한 이온 이동성 질량 분광법 기술

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

정보 베어링 펩토이드의 합성과 시퀀스 지향 동적 공유자 자체 조립

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...