VJ와 HS는 NIH R01 GM094246에서 VJ 로의 지원을 인정합니다. HS는 Clemson University 창의적 탐구 프로그램 및 Clemson University의 광학 재료 과학 및 엔지니어링 기술 센터로부터 창업 자금을 인정합니다. 이 프로젝트는 걸프 코스트 컨소시엄의 학제 간 생물 과학 교육 센터 (NIGMS Grant No. 1 T32GM089657-05)의 켁 센터 (Keck Center)와 일반적인 인간 질병의 번역 연구를위한 시스 손 재단 파운데이션 (Schissler Foundation Fellowship)의 연수 프로그램도 지원했다. 내용은 전적으로 저자의 책임이며 반드시 국립 보건원의 공식 의견을 나타내는 것은 아닙니다.

모든 저자는이 기사의 내용과 경쟁 관계가 없다고 선언합니다.

이 연구에서는 PIE-MFD 단일 분자 FRET 실험을 사용하여 interdye distance를 고정밀 도로 정렬, 교정 및 측정하는 프로토콜을 제시합니다. 모든 기기 파라미터를 신중하게 교정하여 측정 된 거리의 정확도를 높이고 Angstrom 정확도에 도달 할 수 있습니다. 이를 위해 다양한 다차원 히스토그램을 사용하여 추가 특성 분석을위한 모집단을 분석하고 식별합니다. 측정 된 샘플의 안정성을 확인하기 위해 평균 ?...

구조 생물학 연구의 근본적인 목표는 생체 분자 기계의 구조와 기능 사이의 관계를 밝히는 것입니다. 생체 분자 ( 예 : 단백질 및 핵산)의 첫 번째 시각적 인상은 1950 년대에 X 선 결정학 (X-Ray Crystallography) 1 , 2 를 통해 발생했습니다. X 선 결정학은 결정 포장에 의해 제약 된 고해상도 정적 구조 정보를 제공합니다. 따라서 X- 선 구조 모델의 내재적 부동성은 생체 분자의 동적 인 특성을 회피하며 이는 대부분의 생물학적 기능에 영향을 미칩니다 3 , 4 , 5 . 핵 자기 공명 (NMR) 6 , 7 , 8 은 수용액에서 구조 모델을 분석함으로써이 문제에 대한 대안적인 해결책을 제시했습니다. 큰 이점NMR은 구조, 동역학 및 기능 3 , 4 , 5 사이의 본질적인 관계를 명확히하는 데 도움이되는 생체 분자 및 구조적 앙상블의 본질적인 동적 특성을 회복 할 수있는 능력입니다. 그럼에도 불구하고 시료 크기와 많은 양의 시료에 의해 제한되는 NMR은 대형 시스템에 대한 복잡한 표지 전략을 필요로합니다. 그러므로 구조 생물학에서 대안적인 방법을 개발할 긴급한 필요가있다. 역사적으로 FRET가 낮은 정확도 거리 측정을 제공한다는 오해 때문에 Fösterster 공명 에너지 전달 (FRET) 9 는 구조 생물학에서 중요한 역할을하지 못했습니다. 이 프로토콜의 목적은 FRET가 나노 미터 규모의 거리를 결정하는 능력을 재검토하여 이러한 거리가 생체 분자의 구조 모델을 구축하는 데 사용될 수 있도록하는 것입니다. 첫 번째 실험적 검증FRET 효율에 대한 R - 6 의존성은 1967 년 Stryer가 &quot;분광적 인 통치자&quot;로서 다양한 길이의 폴리 프롤린을 측정함으로써 행해졌 다. 유사한 실험이 2005 년에 단일 분자 수준에서 이루어졌다. Polyproline 분자는 이상적이지 않은 것으로 밝혀졌고, 따라서 이중 가닥의 DNA 분자는 나중에 사용되었다. 이것은 정확한 거리 측정을위한 창을 열었고 FRET을 사용하여 생체 분자의 구조적 특성을 확인했습니다. FRET은 interdye 거리 범위가 ~ 0.6-1.3 R 0 인 경우 최적입니다. 여기서 R 0 은 Förster 거리입니다. 단일 분자 FRET 실험에 사용 된 일반적인 형광체의 경우 R 0 은 ~ 50Å입니다. 일반적으로 FRET은 다른 방법보다 구조 및 역학을 해결하고 차별화 할 수있는 많은 장점을 제공합니다.(i) 형광의 궁극적 인 민감성 때문에, 단일 분자 FRET 실험 13 , 14 , 15 , 16 은 개별 구성원의 구조를 직접 세고 동시에 특성화함으로써 이질적인 앙상블을 해결할 수있다. (ii) 앙상블의 동기화가 필요 없기 때문에 단일 분자 FRET 연구에서 복잡한 반응 경로를 직접 해독 할 수 있습니다. (ⅲ) FRET는 생물학적으로 관련된 다양한 역학 관계를 다루면서 10 년 이상 시간 영역의 넓은 영역에 접근 할 수있다. (iv) FRET 실험은 in vitro 및 in vivo의 모든 용액 조건에서 수행 될 수있다. FRET와 형광 현미경의 조합은 생체 내 세포에서 직접적으로 분자 구조와 상호 작용을 연구 할 수있게합니다 15 , 16 , </<html> 17 , 18 , 19 , 고정밀도 20 . (v) FRET은 거의 모든 크기의 시스템 ( 예 : 폴리 프롤린 올리고머 21 , 22 , 23 , 24 , Hsp90 25 , HIV 역전사 효소 26 및 리보솜 27 )에 적용 할 수 있습니다. (vi) 마지막으로, 생체 분자의 모든 차원을 포함하는 거리의 네트워크는 정적 또는 동적 분자 18 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35의 구조 모델을 도출하는데 사용될 수있다 .ref &quot;&gt; 35 , 36 , 37 . 따라서 단일 분자 FRET 분광기를 사용하여 거리가 구속 된 구조 모델링에 사용하기에 충분히 정확한 거리를 유도 할 수 있습니다 26 . 이것은 8 개 차원의 형광 정보 ( 즉, 여기 스펙트럼, 형광 스펙트럼, 이방성, 형광 수명, 형광 양자 수율, 형광 양자 효율 등)를 이용하는 다중 매개 형광 검출 (MFD) 28 , 38 , 39 , 40 , 41 , 거시적 인 시간, 형광 강도 및 형광체 사이의 거리)를 정확하고 정확하게 거리 제한을 제공합니다. 또한, 펄스 인터리빙 된 여기 (PIE)는 MFD(PIE-MFD) (42) 를 사용하여 직접 여기 수용체 형광을 모니터하고 1 : 1 공여체 - 수용체 화학 양롞을 포함하는 샘플에서 발생하는 단일 분자 이벤트를 선택합니다. 일반적인 PIE-MFD 설정은 공 초점 현미경 몸체에 연결된 2 개의 펄스 인터리빙 된 여기 레이저를 사용합니다.이 광자 검출은 서로 다른 스펙트럼 창과 편광 특성의 4 가지 채널로 분할됩니다. 자세한 내용은 그림 1을 참조하십시오. FRET 결과와 일치하는 원자 론적 구조 모델을 얻기 위해 FRET가 계산 방법과 결합되어야한다는 점에 유의해야한다. FRET에서 유도 된 거리를 가진 구조 모델을 구축하는 것은 관련된 방법론을 검토하는 것이 현재의 프로토콜의 목표는 아니다. 그러나 이러한 접근법은 다른 기술 ( 예 : 소각 X 선 산란ing 또는 전자 paramagnetic 공명), 통합 구조 생물학 43 , 44 , 45 , 46 의 분야를 낳았다. 현재의 목표는 구조 생물학에서 정량적 도구로서 FRET의 길을 열어주는 것입니다. 예를 들어,이 방법론은 N- 메틸 -D- 아스 파르 테이트 (NMDA) 수용체의 리간드 - 결합 도메인 (LBD)에서 3 개의 구조 상태를 확인하는데 사용되었다. 궁극적 인 목표는 앞서 언급 한 한계점을 극복하고 고정밀 도로 측정 된 거리를 제공함으로써 생체 분자의 구조적 결정에 사용되는 통합 방법 가운데 FRET를 가져 오는 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="672">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">charcoal</td>
			<td style="width:183px;">Merck KGaA</td>
			<td style="width:119px;">K42964486 320</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">syringe filter</td>
			<td style="width:183px;">Fisherbrand</td>
			<td style="width:119px;">09-719C</td>
			<td>size: 0.20um</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">chambered coverglass</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">155409</td>
			<td>1.5 borosilicate glass, 8 wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">microscope cover glass</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">063014-9</td>
			<td>size: 24X60-1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Nuclease free water</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">148859</td>
			<td>nuclease free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">tween-20</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">28320</td>
			<td>10% solution of Polysorbate 20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (High FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">178124895</td>
			<td>5&#180;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (Low FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177956424</td>
			<td>5&#180;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">donor DNA strand</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177951437</td>
			<td>5&#180; -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">DNA strand (No FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td>5&#180; -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">thermal cycler</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:119px;">E6331000025</td>
			<td>nexus gradient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 488 C5&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A10254</td>
			<td>termed cyan-green fluorophore in the manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 647 C2&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A20347</td>
			<td>termed far-red fluorophore in the manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Rhodamine 110</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83695-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Rhodamine 101</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83694-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">LB Broth, Miller</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1426</td>
			<td>For culture of E. coli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Ampicillin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">A0166</td>
			<td>Used at 100 ug/ml final concentration in selective LB medium to maintain plasmid selection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Tetracyline&#160;</td>
			<td style="width:183px;">Calbiochem</td>
			<td style="width:119px;">58346</td>
			<td>Used at 12.5 ug/ml final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Kanamycin</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP906-5</td>
			<td>Used at 15 ug/ml final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Origami B(DE3) Competent Cells</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:119px;">70837-3</td>
			<td>Competent E. coli cells for expression of protein with disulfide bridges</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Isopropyl-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1755</td>
			<td>For induction of E. coli protein expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">HiTrap Chelating HP</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td style="width:119px;">17-0409-01</td>
			<td>For Large-scale FPLC Purification of His-tagged protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Imidazole</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Ni-NTA Agarose&#160;</td>
			<td style="width:183px;">Qiagen</td>
			<td align="right" style="width:119px;">30210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PD-10 Desalting Column</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td style="width:119px;">17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">AktaPurifier</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td align="right" style="width:119px;">28406264</td>
			<td>FPLC Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Dialysis tubing</td>
			<td style="width:183px;">Spectrum labs</td>
			<td style="width:119px;">132562</td>
			<td>15 kD MWCO 24 mm Flath width, 10 meters/roll</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Dichroics</td>
			<td style="width:183px;">Semrock</td>
			<td style="width:119px;">FF500/646-Di01-25x36</td>
			<td>500/646 BrightLight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">50/50 Beam splitter polarizer</td>
			<td style="width:183px;">Qioptiq Linos&#160;</td>
			<td style="width:119px;">G33 5743 000</td>
			<td>10x10 film polarizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Green pass filer</td>
			<td style="width:183px;">Chroma</td>
			<td style="width:119px;">ET525/50m</td>
			<td style="width:167px;">ET525/50m 25 mm diameter mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Red pass filter</td>
			<td style="width:183px;">Chroma</td>
			<td>ET720/150m</td>
			<td>ET720/150m 25 mm diameter mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Power Meter</td>
			<td style="width:183px;">ThorLabd</td>
			<td style="width:119px;">PM200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">UV-Vis spectrophotometer</td>
			<td style="width:183px;">Varian</td>
			<td style="width:119px;">Cary300Bio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorolog 3 fluorometer</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">FL3-22-R3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorohub TCSPC controller</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">Fluorohub-B</td>
			<td>TCSPC electronics for ensemble measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 485L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">485L</td>
			<td>Blue diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 635L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">635L</td>
			<td>Red diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Olympus IX73 Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">IX73P2F</td>
			<td>Microscope frame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 40 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932200, PMA 40</td>
			<td>Optimized for green detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 50 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932201, PMA 50</td>
			<td>Optimized for ed shifter sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">485nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">640nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-640</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Hydraharp 400 and TTTR acqusition software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">930021</td>
			<td>Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">SEPIA II SLM 828 and SEPIA software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">910028</td>
			<td>Laser driver for picosecond pulses , includes SEPIA software controller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">computer</td>
			<td style="width:183px;">Dell</td>
			<td style="width:119px;">optiplex 7010</td>
			<td>cpu: i7-3770 ram:16GB</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">FRET Positioning and Screening (FPS) software</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">MFD suite</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html</td>
		</tr>
	</tbody>
</table>

high precision fret at single-molecule level for biomolecule structure determination

다중 매개 변수 형광 검출 (MFD) 모드에서 단일 분자 수준에서 Förster 공명 에너지 전달 (FRET)을 사용하여 고정밀 interdye 거리 측정을 수행하는 방법에 대한 프로토콜이 여기에 제시됩니다. MFD는 광 물리 및 실험 결과물을 줄이기 위해 형광의 모든 &quot;차원&quot;의 사용을 극대화하고 단단한 생체 분자에서 ~ 1Å까지의 정확도로 interdye 거리를 측정 할 수 있습니다. 이 방법은 N- 메틸 -D- 아스 파르 테이트 (NMDA) 수용체의 리간드 - 결합 도메인의 3 개의 구조 상태를 확인하여 리간드 결합시 수용체의 활성화를 설명하는데 사용되었다. 알려진 결정학 구조를 실험 측정과 비교할 때, 그들은 더 동적 인 생체 분자에 대해 3Å 미만으로 동의했다. 생체 분자의 전체 차원을 커버하는 일련의 거리 구속을 모으면 동적 생체 분자의 구조 모델을 제공 할 수 있습니다es.

MFD 설정 (레이저 라인 : 60 μW에서 485 nm, 23 μW에서 640 nm, 섹션 5.1)을 사용하는 전형적인 smFRET 실험에서 형광 샘플을 낮은 피코 몰 농도 (10 -12 M = 1 pM)로 희석하고 공진 현미경 (sub-nanosecond laser pulse)은 여기 볼륨 (excitation volume)을 통해 자유롭게 확산되는 라벨 분자를 여기시킨다. 일반적인 공 초점 부피는 &lt;4 펨토텔 (fL)입니다. 그러한 저농도에서는 한 번에 하나의 분...

Watch this Scientific Journal Video about High Precision FRET at Single-molecule Level for Biomolecule Structure Determination at JoVE.com

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

단일 분자 수준에서 고정밀 FRET 실험을위한 프로토콜이 여기에 제시됩니다. 또한,이 방법론은 N- 메틸 -D- 아스 파르 테이트 (NMDA) 수용체의 리간드 결합 도메인에서 3 개의 구조 상태를 확인하는데 사용될 수있다. 정확한 거리를 결정하는 것은 FRET 실험에 기반한 구조 모델을 구축하기위한 첫 번째 단계입니다.

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 ...

The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

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Biochemistry

10.6K 조회수.  Clemson University. The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

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

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

단일 분자의 구조 정보는 고속 나노 측위 시스템을 사용하여 실험을 FRET

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

연구

생화학

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

마그네틱 핀셋으로 G-quadruplexes의 단일 분자 조작

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

높은 처리량, 양적 도트를 사용 하 여 조직 수준에서 선택 된 단백질의 내용의 절대 결정 오 점 분석 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

3 색 단일 분자 무서 워를 사용 하 여 단백질 상호 작용의 상관 관계를 연구 하

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

단일 분자 수준에서 동질성을 라벨의 프로테옴 와이드 정량화

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

Single-Molecule Dwell-Time Analysis of restriction Endonuclease-mediated DNA cleavage(제한 엔도뉴클레아제 매개 DNA 절단의 단일 분자 체류 시간 분석)

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

오픈 소스 smfBox를 사용하여 정확하고 정확한 단일 분자 FRET 측정

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

DNA에 조립되는 EWS-FLI1 응축수의 단일 분자 이미징

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

글리코겐 구조 측정을 위한 간 글리코겐 분자 추출

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

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.