Name: submillisecond conformational changes in proteins resolved by photothermal beam deflection
Uploaded: 2014-02-18T13:00:00
Description: Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

Dette arbejde blev støttet af National Science Foundation (MCB 1.021.831, JM) og J. &amp; E. Biomedical Research Program (Florida Department of Health, JM).

1.. Sample Forberedelser <ol><li> Udføre prøveforberedelse, og alle prøve manipulationer i et mørkt rum for at forhindre en uønsket uncaging. </li><li> Solubilisere DM-nitrophen ((1 - (2-nitro-4 ,5-dimethoxyphenyl) - N, N, N &#39;, N&#39;-tetrakis-[(oxy-carbonyl) methyl] -1,2-ethandiamin) i 50 mM HEPES buffer, 100 mM KCI, pH 7,0 til en endelig koncentration på 400 uM (ε 350nm = 4330 M-1cm-1 9). </li><li> Tilføj CaCl2 fra 0,1 M stamopløsning til at opnå et ønskeligt ...

<ol>
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Det fysiske princip bag photothermal metoder er, at et foto-exciterede molekyle afleder overskydende energi via vibrations afslapning til grundtilstanden, hvilket resulterer i termisk opvarmning af det omgivende opløsningsmiddel 1,12. For opløsningsmidler såsom vand, producerer en hurtig ekspansion volumen (AV th). Spændt-statslige molekyler kan også gennemgå fotokemiske processer, der resulterer i nonthermal volumen ændringer (AV nonth) grundet bindingsspaltning / dannelse og / e...

Foto-termiske metoder såsom fotoakustisk kalorimetri, photothermal stråle deformation (FBF) og forbigående rist kombineret med nanosekund laserexcitation udgør et stærkt alternativ til forbigående optiske spectroscopies for tidsopløste studier af kortlivede mellemprodukter 1,2. I modsætning til optiske teknikker, såsom forbigående absorption og IR-spektroskopi, at overvåge tidsprofilen for absorption ændringer i kromoforen omkring; Photothermal teknikker detektere tidsafhængighed ændringer varme / volumen og er derfor værdifulde værktøjer til undersøgelse tidsprofiler af optisk &quot;tavse&quot; processer. Hidtil fotoakustisk kalorimetri og forbigående rist er med succes blevet anvendt til at studere konformationelle dynamik af foto-inducerede processer, herunder diatomiske ligand migration i globiner 3,4, ligand interaktioner med ilt sensor protein FixL 5, elektron og proton transport i hæm-kobber oxidases 6 and fotosystem II samt foto-isomerisering i rhodopsin 7 og konforme dynamik i cryptochrome 8. For at udvide anvendelsen af ​​photothermal teknikker til biologiske systemer, der mangler en intern kromofor og / eller fluorophor blev PBD teknik kombineret med brugen af ​​bur forbindelse til foto udløser en stigning i ligand / substratkoncentration inden for få mikrosekunder eller hurtigere, afhængigt på bur forbindelse. Denne fremgangsmåde tillader overvågning af dynamik og energetik af strukturelle ændringer i forbindelse med liganden / substrat binding til proteiner, der mangler en intern fluorophor eller chromofor og tidsfrist, som ikke er tilgængelige ved kommercielle stop flow instrumenter. Her en anvendelse af PBD til at overvåge termodynamik buret forbindelse, Ca2 + DM-nitrophen, foto-spaltning samt kinetikken for Ca 2 + associering med det C-terminale domæne af neuronal calcium sensor Downstream Regulatory Element Antagonist Modulator (DREAM) præsenteres. Ca2 + er tilgængeligt udgivet fra Ca 2 + DM-nitrophen inden for 10 mikrosekunder og rebinds en unphotolysed bur med en tidskonstant på ~ 300 mikrosekunder. På den anden side, i nærvær af apoDREAM en yderligere kinetisk forekommer på millisekund målestok observeres og afspejler ligandbinding til proteinet. Anvendelsen af PBD at sonden konformationelle overgange i biologiske systemer er noget begrænset på grund af de instrumentale vanskeligheder, fx besværlig justering af proben og pumpebundt at opnå en stærk og reproducerbar PBD signal. Men en omhyggelig udformning af en instrumentering set-up, en præcis styring af temperaturen, og en omhyggelig tilpasning af sonden og pumpebundt give en konsistent og robust PBD signal, der tillader overvågning af tidsopløst volumen og enthalpi ændringer på en bred tidshorisont fra 10 mikrosekunder til cirka 200 ms. Desuden modifictioner i den eksperimentelle procedure for at sikre påvisning af prøve-og reference-spor under identiske temperatur, buffer sammensætning, optisk celle orientering, laser magt, etc. reducerer den eksperimentelle fejl i afmålte reaktionsvolumener og entalpier.

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			<td height="84" style="height:84px;width:216px;">1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N&#39;,N&#39;-Tetraacetic Acid</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">D-6814</td>
			<td style="width:220px;">DM-nitrophen, cage calcium compound, keep stock solutions in dark to prevent photodissociation,</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td style="width:84px;">Sigma Adrich</td>
			<td>0909C</td>
			<td>HEPES buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium ferricyanide(III)</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">702587</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:84px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:220px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium chromate</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">307831</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">He-Ne Laser Diode 5mW 635nm </td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">54-179</td>
			<td style="width:220px;">use as a probe beam for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Oscilloscope, </td>
			<td style="width:84px;">LeCroy</td>
			<td style="width:119px;">Wave Surfer 42Xs</td>
			<td style="width:220px;">400 MHz bandwith</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nd:YAG laser</td>
			<td style="width:84px;">Continuum</td>
			<td style="width:119px;">ML II</td>
			<td>pump beam for PBD measurments</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M355; Nd:YAG laser mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">47-324</td>
			<td>laser mirror for 355 nm laser line</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M1 and M2; Laser diode mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">43-532</td>
			<td>visilbe laser flat mirror, wavelength range 300-700 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P1 and P2; Iris Diaphragm</td>
			<td style="width:84px;">Edmind Optics</td>
			<td style="width:119px;">62-649</td>
			<td style="width:220px;">pin hole to shape the probe and pum beams</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">L1; bi-convex lens</td>
			<td style="width:84px;">Thorlabs</td>
			<td style="width:119px;">LB1844</td>
			<td style="width:220px;">a lens to focus the probe beam at the detector, EFL 50 mm, wavelength range 350 - 2000 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DM, dichroic mirror</td>
			<td style="width:84px;">Thorlab </td>
			<td style="width:119px;">DMLP505</td>
			<td style="width:220px;">a longpass dichroic mirror with a cutoff wavelength of 505 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F1; Edge filter</td>
			<td style="width:84px;">Andower </td>
			<td style="width:119px;">500FH90-25</td>
			<td>a long pass filter with a cutoff wavelength of 500 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-controlled cuvette holder</td>
			<td style="width:84px;">Quantum Northwest</td>
			<td style="width:119px;">FLASH 300</td>
			<td></td>
		</tr>
	</tbody>
</table>

submillisecond conformational changes in proteins resolved by photothermal beam deflection

Photothermal stråle afbøjning sammen med fotoakustisk kalorimetri og termisk rist hører til familien af ​​photothermal metoder, der overvåger tid profilerede volumen og enthalpi ændringer i lys inducerede konformationsændringer i proteiner på mikrosekund til millisekund-skalaer, der ikke er tilgængelige ved hjælp af traditionel stopper -flow instrumenter. Eftersom de samlede ændringer i mængde og / eller enthalpi probes disse teknikker kan anvendes på proteiner og andre bio-makromolekyler, der mangler en fluorophor og eller en chromophor label. At overvåge dynamik og energetik af strukturelle ændringer, der er forbundet med Ca 2 + binding til calcium transducere sådanne neuronale calcium sensorer, et bur calciumkomponent, DM-nitrophen, er ansat til at foto-udløse en hurtig (τ &lt;20 mikrosekunder) stigning i frit calcium koncentration og den tilhørende volumen og enthalpi ændringer probes hjælp photothermal stråle afbøjning teknik.

Et repræsentativt eksempel på PBD spor for Ca 2 + foto-frigivelse fra Ca2 + DM-nitrophen er vist i figur 3.. Den hurtige fase svarer til foto-spaltning af Ca 2 + DM-nitrophen og Ca 2 + befrielse, mens den langsomme fase afspejler Ca 2 + binding til nonphotolysed bur. Plottet af prøven PBD amplitude for hurtig og langsom fase skaleret til amplitude referenceforbindelse som en funktion af temperaturen afhang faktor [Cp ρ / (dn / dt)] fa...

Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Her rapporterer vi en anvendelse af photothermal stråle afbøjning teknik i kombination med et bur calciumkomponent, DM-nitrophen, overvåge mikrosekund og millisekund dynamik og energetik af strukturelle ændringer i forbindelse med calcium association til en neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator .

Submillisecond konformationsændringer i Proteiner løst ved Photothermal Beam Nedbøjning

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

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