VJ ve HS, NIH R01 GM094246&#39;dan VJ&#39;ye verilen desteği onaylar. HS, Clemson Üniversitesi Yaratıcı Sorgulama Programı ve Clemson Üniversitesi&#39;ndeki Optik Malzeme Bilimi ve Mühendisliği Teknolojileri Merkezi&#39;nden yeni teşebbüs kredilerini onayladı. Bu proje aynı zamanda Körfez Sahil Konsorsiyumu için Keck Merkezi&#39;nden (NIGMS Grant No.1 T32GM089657-05) Interdisciplinary Bioscience Training için bir eğitim fakültesi ve DD için Ortak İnsan Hastalıklarının Translasyonel Çalışmaları için Schissler Vakfı Topluluğu tarafından da desteklendi. İçerik sadece yazarların sorumluluğundadır ve Ulusal Sağlık Enstitüleri&#39;nin resmi görüşlerini temsil etmez.

Tüm yazarlar, bu maddenin içeriğiyle rekabet eden mali çıkarlarının olmadığını beyan eder.

Bu çalışmada, PIE-MFD tek moleküllü FRET deneyleri kullanılarak, boyalar arası mesafeleri yüksek hassasiyetle hizalamak, kalibre etmek ve ölçmek için protokol sunulmuştur. Tüm enstrüman parametrelerini titizlikle kalibre ederek, ölçülen uzaklıkların doğruluğunu artırabilir ve Angstrom doğruluğuna erişebilirsiniz. Bunu yapmak için, daha fazla karakterizasyon için popülasyonları analiz etmek ve tanımlamak için çeşitli çok boyutlu histogramlar kullanılır. Ölçülen numunelerin kararlıl...

Yapısal biyoloji çalışmalarının temel amacı, biyomoleküler makinelerin yapısı ve işlevi arasındaki ilişkiyi ortaya çıkarmaktır. Biyomoleküllerin ( örn., Proteinler ve nükleik asitler) ilk görsel izlenimi 1950&#39;lerde X-ışını kristalografisi 1 , 2 ile oluştu. X-ışını kristalografisi, kristal ambalaj ile sınırlandırılmış yüksek çözünürlüklü, statik yapısal bilgiler sağlar. Bu nedenle, X-ışını yapısal modellerinin doğasında olan hareketsizliği, biyomoleküllerin dinamik doğasından kaçınır; bu, çoğu biyolojik fonksiyonları etkileyen bir faktördür 3 , 4 , 5 . Nükleer manyetik rezonans (NMR) 6 , 7 , 8 sulu solüsyonlarda yapısal modelleri çözerek problemin alternatif bir çözümünü sağlamıştır. Büyük bir avantajNMR, yapısı, dinamikleri ve fonksiyonu arasındaki gerçek ilişkileri netleştirmeye yardımcı olan biyomoleküllerin ve konformasyonel toplulukların intrensek dinamik doğasını kurtarma yeteneğidir. 3 , 4 , 5 . Bununla birlikte, NMR, numune büyüklüğü ve büyük miktarlarda numune ile sınırlı olduğundan, daha büyük sistemler için karmaşık etiketleme stratejileri gerektirir. Bu nedenle, yapısal biyolojide alternatif yöntemler geliştirmek için acilen bir ihtiyaç vardır. Tarihsel olarak, Förster rezonans enerji transferi (FRET) 9 , yapısal biyolojide önemli bir rol oynamadı FRET&#39;in düşük doğruluklu mesafe ölçümleri sağladığı yanılgısından dolayı. Bu protokolün amacı, FRET&#39;in nanometre ölçeğindeki mesafeleri belirleme yeteneğini tekrar gözden geçirmektir; bu mesafeler, biyomoleküllerin yapısal modellerini oluşturmak için kullanılabilir. İlk deneysel doğrulamaFRET verimliliğine R - 6 bağımlılığının bağımlılığı, 1967&#39;de Stryer tarafından 10 &quot;spektroskopik cetvel&quot; olarak çeşitli uzunluklarda poliprolinlerin ölçülmesi ile gerçekleştirildi. Benzer bir deney, 2005 yılında tek molekül seviyesinde başarılmıştır 11 . Poliprolin molekülleri ideal olmayan oldukları ortaya çıkmış ve bu nedenle çift sarmallı DNA molekülleri daha sonra kullanılmıştır 12 . Bu, kesin uzaklık ölçümleri için pencereyi açtı ve biyomoleküllerin yapısal özelliklerini tanımlamak için FRET&#39;in kullanılması fikrini verdi. FRET, boyanacak mesafe aralığı ~ 0.6-1.3 R 0 arasındayken optimaldir, burada R 0 Förster mesafesidir. Tek moleküllü FRET deneylerinde kullanılan tipik fluoroforlar için R 0 ~ 50 A&#39;dır. Tipik olarak FRET, yapıları ve dinamikleri çözme ve ayırma becerisi bakımından diğer yöntemlere göre birçok avantaj sunmaktadırHeterojen sistemler: (i) Floresansın nihai hassasiyetinden dolayı, tek moleküllü FRET deneyleri 13 , 14 , 15 , 16 heterojen toplulukları bireysel üyelerin yapılarını doğrudan sayarak ve aynı anda karakterize ederek çözümleyebilir. (Ii) Kompleks reaksiyon yolakları tek moleküllü FRET çalışmalarında doğrudan çözülebilir, çünkü bir toplulukta senkronizasyona ihtiyaç yoktur. (Iii) FRET, biyolojik olarak ilgili dinamikleri içeren geniş bir yelpazede, zaman içinde 10 yılı aşan geniş bir zaman alanlarına erişebilir. (Iv) FRET deneyleri, in vitro olarak ve in vivo olarak herhangi bir çözelti koşulunda gerçekleştirilebilir. FRET&#39;in flüoresans mikroskobu ile kombinasyonu, canlı hücrelerdeki moleküler yapıların ve etkileşimlerin incelenmesine olanak tanır 15 , 16 , </<html> &gt; 17 , 18 , 19 , hatta yüksek hassasiyetle 20 . (V) FRET , neredeyse her boyutta ( örneğin, poliprolin oligomerler 21 , 22 , 23 , 24 , Hsp90 25 , HIV revers transkriptaz 26 ve ribozomlar 27 ) uygulanabilir. (Vi) Son olarak, biyomoleküllerin tüm boyutlarını içeren mesafeler ağı, statik veya dinamik moleküllerin 18 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ,Ref &quot;&gt; 35 , 36 , 37 . Bu nedenle, tek moleküllü FRET spektroskopisi, mesafeye göre sınırlandırılmış yapısal modellemede kullanılabilecek kadar hassas mesafeler türetmek için kullanılabilir. Bu, flüoresans bilgisinin sekiz boyutunu ( yani uyarma spektrumu, floresans spektrumu, anizotropi, floresans ömrü, floresan kuantum verimi, Makroskopik zaman, flüoresans yoğunlukları ve flüoroforlar arasındaki mesafe) doğru ve tam olarak mesafe sınırlamaları sağlar. Buna ek olarak, darbeli araya sokulan uyarılma (PIE), MFD(PIE-MFD) 42 ile direkt uyarım akseptörünün flüoresansını izlemek ve 1: 1 donör-toplayıcı stokiyometri içeren numunelerden kaynaklanan tek molekül olaylarını seçmek için kullanılmıştır. Tipik bir PIE-MFD kurulumu, foton algılamanın farklı spektral pencerelerde ve kutuplama özelliklerinde dört farklı kanala ayrıldığı bir konfokal mikroskop gövdesine bağlı iki palslı araya sokulmuş uyarma lazerleri kullanır. Daha fazla ayrıntı Şekil 1&#39;de bulunabilir . FRET&#39;in 26 , 30 FRET sonuçları ile uyumlu atomistik benzeri yapısal modelleri elde etmek için FRET&#39;in hesaplama yöntemleriyle birleştirilmesi gerektiğini unutmamak önemlidir. Mevcut protokolün amacı, FRET kaynaklı mesafelerde yapısal modeller oluşturmak için ilgili metodolojiyi gözden geçirmek değildir. Bununla birlikte, bu yaklaşımlar diğer tekniklerle ( örn. Küçük açılı X ışını saçılmasıIng ya da elektron paramanyetik rezonans), bütünleyici yapısal biyoloji alanını doğuruyor 43 , 44 , 45 , 46 . Halihazırdaki amaç yapısal biyolojide nicel bir araç olarak FRET&#39;in yolunu açmaktır. Bir örnek olarak, bu metodoloji, N-metil-D-aspartat (NMDA) reseptörünün ligand bağlama alanındaki (LBD) üç konformasyonel durumu tanımlamak için kullanıldı. Nihai amaç, belirtilen sınırlamaları aşmak ve FRET&#39;i, yüksek hassasiyette ölçülen mesafeler sağlayarak biyomoleküllerin yapısal belirlenmesi için kullanılan bütünleştirici yöntemler arasında getirmektir.

<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

Çok parametreli flüoresan algılama (MFD) modunda tek molekül seviyesinde Förster rezonans enerji transferi (FRET) kullanılarak yüksek hassasiyetli boyacılar arası mesafe ölçümlerinin nasıl yapılacağı üzerine bir protokol burada sunulmuştur. MFD, fotofizik ve deneysel eserleri azaltmak için flüoresanın tüm &quot;boyutlarını&quot; maksimize eder ve rijid biyomoleküllerde ~ 1Å kadar bir doğrulukla boyarmadde mesafe ölçümünü sağlar. Bu yöntem, ligand bağlanması üzerine reseptörün aktivasyonunu açıklamak için N-metil-D-aspartat (NMDA) reseptörünün ligand bağlama alanının üç konformasyonel durumunun tanımlanması için kullanılmıştır. Bilinen kristalografik yapıları deneysel ölçümlerle karşılaştırırken, daha dinamik biyomoleküller için 3 A&#39;dan küçük bir alan üzerinde anlaşmışlardır. Biyomoleküllerin tüm boyutlarını kapsayan bir dizi mesafe sınırlamaları toplamak dinamik biyomolekülün yapısal bir modelini mümkün kılabilires.

Bir MFD kurulumu (lazer çizgileri: 60 μW&#39;da 485 nm ve 23 μW&#39;da 640 nm, bölüm 5.1) kullanan tipik smFRET deneylerinde, floresans numunesi düşük pikomolar konsantrasyonda ( 10-12 M = 1 pM) seyreltilir ve Bir alt-nanosaniyelik lazer darbesinin uyarı hacmi boyunca serbestçe dağılmış etiketli molekülleri uyaran bir konfokal mikroskopta. Tipik bir konfokal hacim &lt;4 femtolitre (fL) &#39;dir. Düşük konsantrasyonlarda, tek seferde tek bir molekül tespit ed...

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

Tek molekül seviyesinde yüksek hassasiyetli FRET deneyleri için bir protokol burada sunulmuştur. Buna ek olarak, bu metodoloji, N-metil-D-aspartat (NMDA) reseptörünün ligand bağlama alanındaki üç konformasyonel durumu tanımlamak için kullanılabilir. Kesin mesafelerin belirlenmesi FRET deneylerine dayanan yapısal modellerin oluşturulmasına yönelik ilk adımdır.

Biyomolekül Yapısının Belirlenmesi İçin Tek Molekül Seviyesinde Yüksek Hassasiyetteki FRET

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

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

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

Research

JoVE Journal

Biochemistry

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

Video: 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.

Tek molekülün yapısal bilgiler Hızlı Nano konumlandırma Sisteminin Kullanılması Deneyler FRET

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

Biyokimya

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.

Yüksek işlem hacmi, mutlak nicel nokta kullanarak doku düzeyleri, seçili bir Protein içeriği tayini Blot Analizi (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.

Üç renkli tek molekül FRET Protein etkileşimleri korelasyon çalışma için kullanma

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.

Proteom çapında Quantification maddelerinin homojenliği tek molekül düzeyinde etiketlenmesi

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.

Restriksiyon endonükleaz aracılı DNA bölünmesinin tek moleküllü bekleme süresi analizi

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.

Açık Kaynaklı smfBox kullanarak Hassas ve Doğru Tek Moleküllü FRET Ölçümleri Yapma

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

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.

Glikojen Yapısının Belirlenmesi için Karaciğer Glikojen Moleküllerinin Ekstraksiyonu

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.

Tek Moleküllü FRET Kullanarak Membran Reseptörlerinin Konformasyonel Dinamiğinin Görselleştirilmesi

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

Kriyo Numune Hazırlamayı İyileştirmek için CryoEM Izgaralarının Tek Katmanlı Grafen ile Kaplanması

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Yazar Spotlight: Mikroskopi Izgaralarında Grafen Tek Tabaka Kullanarak CryoEM Numune Hazırlamanın Geliştirilmesi 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

Yüksek Çözünürlüklü Yapı Belirleme için Tek Katmanlı Grafenin Kriyo-Elektron Mikroskobu Izgaralarına Uygulanması

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...