This work is supported by the US National Institutes of Health (R01GM111452, Y.W., S.X.). Y. W. acknowledges support from the Welch Foundation (E-1721).

1. Preparation of the ribosome complexes
<ol>
	<li>Make 1,000 mL of TAM10 buffer, which consists of 20 mM tris-HCl (pH 7.5), 10 mM Mg (OAc)2, 30 mM NH4Cl, 70 mM KCl, 5 mM EDTA, 7 mM BME (2-mercaptoethanol), and 0.05% Tween20.</li>
	<li>Prepare the five mixtures listed in Table 1. 
	NOTE: The ribosome was from the MRE600 strain11. EF-Tu: elongation factor thermo unstable. EF-Ts: elongation factor thermo stable. GTP: guanosine triphosphate. PEP: phospho(...

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In our dual ruler assay, the magnetic beads play two essential roles. First, they serve as the force transducers because the centrifugal force is proportional to their buoyant mass. Second, the beads are signal carriers detected by an atomic magnetometer, which is currently the most accurate magnetic sensor. Combining mechanical manipulation and magnetic detection, the FIRMS technique is able to resolve a large number of molecular interactions based on their critical dissociation forces, which is the basis of the DNA rul...

Biomolecular displacement is a fundamental parameter in studying the mechanism of the related biological functions. One particular example is the ribosome translocation1,2, during which the ribosome moves by exactly three nucleotides (nt) on the messenger RNA (mRNA) normally, and by one, two, or other numbers of nt except three in the case of frameshifting. Therefore, a molecular ruler system single-nt resolution is required to distinguish the different step sizes. A greater challenge is to probe the ribosome movement on both the entrance and exit sides. In other words, only with a dual ruler system will we be able to reveal whether the mRNA is smoothly threaded through the ribosome, or there are intermediate steps in which the two sides have different step sizes leading to a kinked or looped mRNA conformation inside the ribosome.
Several methods have been developed to address the first challenge of resolving different steps on the exit side of the ribosome (the 3&#39;&#160;end of the mRNA). The dual luciferase assay resolves the different reading frames by measuring the ratios of the resulting different proteins3,4. It is only applicable for the 3&#39;&#160;end of the mRNA and thus insufficient to provide a complete picture of translocation. Mass spectrometry can analyze the different peptide fragments as the consequence of the corresponding code rearrangements5. But it cannot pinpoint to how many nt the ribosome moves on the mRNA. The toe-printing assay is another common method that uses a reverse transcriptase primed at the 3&#39;-distal end to transcribe the mRNA toward the ribosome6. However, it is not applicable for the 5&#39;&#160;end of mRNA that is entering the ribosome. Other techniques, including single molecule approaches and fluorescence methods7, are difficult to achieve single-nt resolution.
We have developed the dual DNA ruler assay that can uniquely determine both the entrance and exit positions of the uncovered mRNA in ribosome-mRNA complexes. &#160;The ruler DNAs are DNA oligomers that form duplexes of certain numbers of basepairs (bp) with the mRNA uncovered by the ribosome, regardless of which end of the mRNA. The bp numbers then precisely reveal the ribosome position on the mRNA during translocation. The bp numbers of the duplexes are determined by their critical dissociation forces obtained from force-induced remnant magnetization spectroscopy (FIRMS)8. With 2-3 pN force uncertainty, the critical forces are sufficient to offer single-nt resolution. By implementing a linker molecule on the DNA rulers, the sterically hindered side of the mRNA by the ribosome can be probed. Different ribosomal displacements can thus be accurately resolved. We have successfully revealed a unique looped conformation of mRNA trapped by antibiotics during translocation9, and resolved different reading frames that coexisted on a slippery mRNA sequence10. This article describes the details of the dual ruler assay, which include preparation of the ribosome complexes, surface functionalization of the glass slides, immobilization of the ribosome complexes and their hybridization with magnetically labeled DNA ruler molecules, magnetic detection, and force spectrum analysis by FIRMS.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:1412px;" width="1411">
	<colgroup>
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			<td height="21" style="height:21px;width:217px;">Styrene Strip</td>
			<td style="width:239px;">City of Industry</td>
			<td style="width:161px;">MS-861</td>
			<td style="width:795px;"></td>
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			<td style="width:239px;">Evaporated Coatings</td>
			<td style="width:161px;"></td>
			<td>60.0 &#215; 4.0 &#215; 0.3 mm3</td>
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			<td height="21" style="height:21px;width:217px;">Acetic acid</td>
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			<td style="width:161px;">A6283-500ML</td>
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			<td height="21" style="height:21px;width:217px;">3-Aminopropyltriethoxysilane</td>
			<td style="width:239px;">UCT specialties</td>
			<td style="width:161px;">21400088</td>
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			<td height="21" style="height:21px;width:217px;">mPEG-SVA</td>
			<td style="width:239px;">Laysan Bio</td>
			<td style="width:161px;">154-82</td>
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			<td height="21" style="height:21px;width:217px;">Biotin-PEG-SVA</td>
			<td style="width:239px;">Laysan Bio</td>
			<td style="width:161px;">152-84</td>
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			<td height="21" style="height:21px;width:217px;">Sodium bicarbonate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">S5761-500G</td>
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			<td height="21" style="height:21px;width:217px;">Epoxy glue</td>
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			<td style="width:161px;">31345</td>
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			<td style="width:239px;">ThermoFisher</td>
			<td style="width:161px;">434301</td>
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			<td height="21" style="height:21px;width:217px;">Fusidic Acid</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">F0756-1G</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Neomycin Sulfate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">1458009</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Viomycin Sulfate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">1715000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Hygromycin</td>
			<td style="width:239px;">invitrogen</td>
			<td style="width:161px;">10687-010</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Tris-HCl</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">T5941-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Magnesium acetate</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M5661-50G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Ammonium chloride</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">A9434-500G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Potassium chloride</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P9333-500G</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">EDTA</td>
			<td style="width:239px;">GIBCO</td>
			<td style="width:161px;">774750</td>
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			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">M6250-500ML</td>
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			<td height="21" style="height:21px;width:217px;">Tween20</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P1379-250ML</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">GTP</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">G8877-100MG</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">PEP</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P7127-100MG</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Pyruvate Kinase</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">P1506-5KU</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Sucrose&#160;</td>
			<td style="width:239px;">Millipore Sigma</td>
			<td style="width:161px;">S7903-5KG</td>
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			<td height="21" style="height:21px;width:217px;">Dynabeads M-280 Streptavidin</td>
			<td style="width:239px;">ThermoFisher</td>
			<td style="width:161px;">11205D</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">mRNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">133899727</td>
			<td>5&#8242;-Bio- CAA CUG UUA AUU AAA UUA AAU UAA AAA GGA AAU AAAA AUG UUU AAU UUU UUA GGG CGC AAU CUA CUG CUG AAC UC-3&#8242;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">157468630</td>
			<td>3&#697;- TAA TTT AAT TTA ATT TTT CGA AAU AT50/TEGBio/-5&#697;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">164845370</td>
			<td>3&#697;-AAT TTA ATT TTT CCT TTA AAA AT50/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">157468628</td>
			<td>3&#697;-AAA ATC CCG CGT TAG AAC UGG GG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">163472705</td>
			<td>3&#697;-CCG CGT TAG ATG ACG AGA ACG GG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678130</td>
			<td>3&#697;-AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678131</td>
			<td>3&#697;-T AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678132</td>
			<td>3&#697;-TT AGA TGA CGA CTT CTC GGG/TEGBio/-5&#697;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">DNA Oligo</td>
			<td style="width:239px;">Integrated DNA Technologies</td>
			<td style="width:161px;">138678133</td>
			<td>3&#697;-GTT AGA TGA CGA CTT CTC GGG/TEGBio/-5&#8217;&#160;</td>
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			<td height="21" style="height:21px;width:217px;">Centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:161px;">5427R</td>
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			<td height="21" style="height:21px;width:217px;">Micro Ultracentrifuge</td>
			<td style="width:239px;">Hitachi</td>
			<td style="width:161px;">CS150FNX</td>
			<td></td>
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			<td height="21" style="height:21px;width:217px;">Vortex mixer</td>
			<td style="width:239px;">VWR</td>
			<td style="width:161px;">VM-3000</td>
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			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR530</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Lock-in Amplifier</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">SR830</td>
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			<td height="21" style="height:21px;width:217px;">Laser</td>
			<td style="width:239px;">Newport</td>
			<td style="width:161px;">TLB-6918-D</td>
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			<td height="21" style="height:21px;width:217px;">Function generator</td>
			<td style="width:239px;">Stanford Research Systems</td>
			<td style="width:161px;">DS345</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Photo detectors</td>
			<td style="width:239px;">Thorlabs</td>
			<td style="width:161px;">DET36A</td>
			<td></td>
		</tr>
	</tbody>
</table>

dual dna rulers to study the mechanism of ribosome translocation with single-nucleotide resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Figure 1 shows the detection scheme and photographs of the major components. Magnetic detection is achieved by an atomic magnetometer using the scanning scheme (Figure 1A)13. The sample is placed on a rod mounted on a linear motor. The motor transports the sample to the atomic sensor inside a magnetic shield, then back to the original site for unloading. The atomic magnetometer detects the magnetic signal during the sampl...

Watch this Scientific Journal Video about Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution at JoVE.com

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

The dual DNA ruler assay can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements. Our dual DNA ruler assay is currently the only method that can objectively probe both the entrance and exit sides of mRNA in the ribosome complexes with single-nucleotide resolution. Similar methods are also applied to measure DNA binding strength and selectivity of chemotherapeutical drugs.Visual demonstration of the method is a good chance for us to intuitively show the advantages of our technique and help other researchers to further develop it into more applications. To begin, make a plastic sample well with a styrene strip by drilling a cube in the center with dimensions of four millimeters by three millimeters by two millimeters. Then, using epoxy, glue a piece of biotin-coated glass, of approximately five millimeters long, to the bottom surface.Add 20 microliters of 0.25 milligram per milliliter streptavidin aqueous solution into the sample well, and incubate at room temperature for 40 minutes. Then rinse the sample well twice with TAM10 buffer. To immobilize the ribosome complexes without antibiotics, first remove the buffer from the sample well, and add 20 microliters of 0.1 micromolar MF-Pre or MF-Post ribosome complex into the sample well.Incubate at 37 degrees Celsius for one hour, and then rinse once with TAM10 buffer. The ribosome complex now binds with the streptavidin on the surface via the five-prime end biotin on the mRNA. For the experiment using antibiotics, incubate nine microliters of the MF-Pre complex with one microliter of four millimolar neomycin, 37 degrees Celsius for 10 minutes.Then combine in a tube 0.6 microliters of 60 micromolar EFG, 0.8 microliters of 100 micromolar GTP, 0.8 microliters of 100 millimolar PEP, 0.3 microliters of 1.3 milligram per milliliter pyruvate kinase, 6.43 microliters TAM10 buffer, and one microliter of five millimolar fusidic acid. Incubate at 37 degrees Celsius for 20 minutes. Then combine the two mixtures, and incubate at 37 degrees Celsius for an additional five minutes.Carry out the other antibiotics experiments similarly, with concentrations of 0.2 millimolar viomycin, 0.4 millimolar hygromycin B, and 0.25 millimolar fusidic acid. For frame shifting study, repeat the incubation for the MFNF-Pre complexes involving the slippery motif, U6A. Include antibiotics of fusidic acid plus neomycin.Sufficient time is necessary to ensure the completion of the hybridization process. It is also advised to use the TAM10 buffer with actual one mole sodium chloride to maintain a homosteady system. After incubation, remove buffer from the sample well.Add 20 microliters of one micromolar biotinylated probing DNA strand, and incubate at room temperature overnight. In the morning, rinse the formed DNA/mRNA duplex once with TAM10 buffer. Remove the buffer from the sample well.Then add 20 microliters of 0.5 milligrams per milliliter streptavidin-coated magnetic beads into the sample well, and incubate at room temperature for two hours. After that, carefully insert the sample well into a holder, and place it in a centrifuge to remove the free magnetic particles from the surface at 84 times g for five minutes. Turn on the laser.Then press the power button. Adjust the sensitivity to 500 millivolts, and wait for about two hours to warm up and stabilize the atomic magnetometer. To set up the atomic magnetometer, run the instrument control software, and set up the Motor moving mode to Noise and default position to zero.Press Lock on the front panel. Adjust the sensitivity back to 200 millivolts. Adjust the current and voltage of the laser, and find the proper resonance peak and signal-to-noise level for the best signal resolution.Press Sweep on the front panel. Make sure the amplitude-to-width ratio is above 0.5 and the phase value is smaller than five degrees with width less than 170 hertz. Plug in the output of another SR530 lock-in amplifier to the feedback of the laser to lock its frequency.Plug in function generator ATF20B to input a square wave of 500 millivolts peak to peak, 100 millihertz as the reference signal for converting current to magnetic signal. Then unplug the function generator. Set up the Motor moving mode to Two-way and default position to 260 millimeters.Check the status of the temperature controller, to ensure the temperature is at approximately 37 degrees Celsius, of the atomic sensor. Check the stability of the whole system twice by directly running the program without loading samples to measure the signals of the empty sample holder. To magnetize the samples, gently use tweezers to take the sample out of the holder, and place on the magnetization stage for two minutes.Then put the sample back into the sample holder, and centrifuge at 335.4 times g for 20 minutes. Remove the nonspecifically-bound magnetic particles. Next load the sample onto the motor with tweezers.Click Lock on the front panel to run the program. Meanwhile use tweezers to load another sample into the holder, and place it into the centrifuge. Make the coated glass side face the center of the centrifuge, and start the centrifugation at 335.4 times g for four minutes.When the motor comes back to zero, after approximately five minutes, click Save on the front panel. Carefully use tweezers to transfer one sample from the motor to the centrifuge holder. Apply a stronger force by increasing the centrifugal speed by 100 rpm or a similar step size.Exchange the sample for the centrifuge. Process alternately to gradually increase the force each time. A complete force spectrum is obtained after 10 to 12 data points.When all planned experiments are finished, turn off the equipment, proceeding in the opposite order as it was turned on. Remove the samples from the holder, and immerse them in ethanol for cleaning and future use. Clean up the sample holder with acetone in case of magnetic bead contamination.In this protocol, the ribosome complex was immobilized on the surface, via the five-prime end of the mRNA, and made both the five-prime and three-prime side easily accessible for the probing DNA rulers. When the linker for the five-prime side was longer than 50 thymine, a strong magnetic signal was detected, indicating successful formation of DNA/mRNA duplexes. By varying the number of nucleotides on the DNA, the correlation of dissociation force to duplex length was achieved for the three-prime and five-prime sides, respectively.The results of normal translocation from MF-Pre to MF-Post, in the absence of antibiotics, show that the ribosome moved by three nucleotides toward the three-prime end on both sides. MF-Pre carried a vagrant fMet-tRNA and phenylalanine-tRNA at the P and A sites, respectively, while MF-Post carried fMet-tRNA and phenylalanine-tRNA at the E and P sites, respectively, with a vacant A site. However, when both fusidic acid and adiomycin antibiotics were present, the ribosome moved by only one nucleotide at the five-prime side but two nucleotides at the three-prime side.After washing away the antibiotics, normal translocation occurred, as evidenced by three nucleotide movements from both the five-prime and three-prime sides. When magnetizing the sample, the coated surface should approach and leave the magnet vertically. And removing the nonspecifically-bound magnetic particles is very important for data accuracy and quality.Our technique provides a high resolution and a generally applicable way to investigate molecular displacement of nucleic acid which is widely encountered in molecular biology.

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JoVE Journal

Biochemistry

6.2K ビュー.  University of Houston. 二重DNA定規アッセイは、リボソーム転座の機械学的洞察を提供し、他の核酸変位を探査することができる。当社の二重DNA定規アッセイは、現在のところ、単一ヌクレオチド分解能を有するリボソーム複合体中のmRNAの入口側と出口側の両方を客観的に探査できる唯一の方法です。同様の方法は、DNA結合強度および化学療法薬の選択性を測定するためにも適用される。...

ビデオ: Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid...

single molecule fluorescence energy transfer study of ribosome protein synthesis

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not...

methods to increase the sensitivity of high resolution melting single nucleotide polymorphism genotyping in malaria

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting...

iclip - transcriptome-wide mapping of protein-rna interactions with individual nucleotide resolution

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding...

proofreading and dna repair assay using single nucleotide extension and maldi-tof mass spectrometry analysis

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

genotyping single nucleotide polymorphisms in the mitochondrial genome by pyrosequencing

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

de novo identification of actively translated open reading frames with ribosome profiling data

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome...

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

a scalable method to study neuronal survival in primary neuronal culture with single-cell and real-time resolution

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed...

application of stopped-flow kinetics methods to investigate the mechanism of action of a dna repair protein

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA...

combining single-molecule manipulation and imaging for the study of protein-dna interactions

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped...

atomic force microscopy investigations of dna lesion recognition in nucleotide excision repair

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are...

hybrid ensemble and single-molecule assay to image the motion of fully reconstituted cmg

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical...

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

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nonradioactive assay to measure polynucleotide phosphorylation of small nucleotide substrates

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA...

single-molecule super-resolution imaging of phosphatidylinositol 4,5-bisphosphate in the plasma membrane with novel fluorescent probes

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer...

visualizing single-molecule dna replication with fluorescence microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real...

deciphering molecular mechanism of histone assembly by dna curtain technique

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+...

optical recording of suprathreshold neural activity with single-cell and single-spike resolution

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the...

application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression...

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

using three-color single-molecule fret to study the correlation of protein interactions

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

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...

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.