The experiments were performed at the Light Microscopy Technology Platform (LiMiTec) of the Faculty of Biology, Bielefeld University. This work has been funded by Bielefeld University.

NOTE: For the following protocol, transient transfection of protoplasts was performed, as described previously12. A brief description is given below.
1. Transient transfection of protoplasts
<ol>
	<li>Cut ~4 g of healthy leaves of Arabidopsis thaliana ecotype Columbia into 1 mm slices and transfer them to 20 mL of enzyme solution (1.5% cellulase; 0.4% macerozyme; 0.1% bovine serum albumin Fraction V; 0.4 M mannitol; 20 mM KCl; 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7; 10 mM CaCl2).</li>
	<li>Vacuum-infiltrate the leaf slices followed by an incubation with agitation for 2 h at room temperature. Harvest the cells by centrifugation for 3 min at 100 &#215; g.</li>
	<li>Wash the protoplasts with W5-solution (154 mM NaCl; 125 mM CaCl2; 5 mM KCl; 2 mM MES, pH 5.7) and resuspend them in MMG solution (0.4 M mannitol; 15 mM MgCl2; 4 mM MES, pH 5.7).</li>
	<li>Perform the transfection in an 8-well slide by osmotic shock in the presence of polyethyleneglycol (PEG) 4000. Mix 20 &#181;L of the protoplast suspension with 5 &#181;L of plasmid DNA (5 &#181;g/&#181;L) and 25 &#181;L of PEG solution (0.2 M mannitol, 0.1 M CaCl2, 40% PEG 4000).</li>
	<li>Reverse the osmotic shock by gentle readjustment of the osmotic conditions. 
	&#8203;NOTE: Besides the sample of interest, the expression of donor alone and acceptor alone is required to determine the spectral bleed-through of the donor and the acceptor, respectively. A fusion protein of the donor and the acceptor must be expressed too for calibration purposes. The fluorescent protein expression was under the control of a cauliflower mosaic virus 35S promoter (pCaMV35S). For all measurements, two confocal laser scanning microscopes (LSM1 and LSM2) were used. LSM1 has two types of detectors: for FRET measurements, the donor signal was detected by a GaAsP-detector, while FRET and acceptor emission were recorded with a photomultiplier. LSM2 has two photomultipliers, which were used for the detection of donor, FRET, and acceptor emission.</li>
</ol>
2. Laser-adjustment
NOTE: Here, 458 nm and 514 nm lines of an argon-ion laser have been applied for FRET analysis between enhanced cyan fluorescent protein (ECFP)- and enhanced yellow fluorescent protein (EYFP)-labeled proteins. For reproducible data acquisition, both lines were adjusted to similar intensity. This was achieved by either a transmission photomultiplier or the reflection mode.
<ol>
	<li>Laser adjustment with a transmission photomultiplier
	<ol>
		<li>Use an empty well for adjustment.</li>
		<li>Choose line-scanning mode and histogram view.</li>
		<li>Decrease the laser intensity to the minimum, and adjust the detector gain to detectable background noise.</li>
		<li>Increase the laser intensity in steps of 0.5% and record the corresponding signal.</li>
		<li>Apply the routine for both laser lines.</li>
	</ol>
	</li>
	<li>Laser adjustment with reflection mode
	<ol>
		<li>Use an empty well for adjustment.</li>
		<li>Apply a reflection filter, switch on the reflection mode, if available.</li>
		<li>Ensure that the detector wavelength range covers the wavelength of the laser.</li>
		<li>Choose the line-scanning mode and histogram view.</li>
		<li>Decrease the laser intensity to the minimum, and adjust the detector gain to detectable background noise.</li>
		<li>Move the objective to the lowest position.</li>
		<li>Move the objective up until the reflection of the coverslip is visible.</li>
		<li>Increase the laser intensity in steps of 0.5% and record the corresponding signal.</li>
		<li>Apply the routine for both laser lines.</li>
	</ol>
	</li>
	<li>Data evaluation
	<ol>
		<li>Tabulate the data and sort the data by signal intensities.</li>
		<li>Plot the signal intensities against the relative laser power.</li>
		<li>Choose laser intensities that result in similar signal intensity.</li>
	</ol>
	</li>
</ol>
3. Adjustment of photomultipliers
NOTE: After laser adjustment, the photomultipliers were adjusted to individual gains to obtain similar sensitivity. This calibration was done with the 514 nm laser line, which is in the center of the wavelength range of interest.
<ol>
	<li>Use an empty well for adjustment.</li>
	<li>Apply a reflection filter, and switch to reflection mode if available.</li>
	<li>Ensure that the detector wavelength range covers the wavelength of the laser (514 nm).</li>
	<li>Choose the line scanning mode and histogram view.</li>
	<li>Decrease detector gain to half the maximum, and adjust the laser intensity to detectable background noise.</li>
	<li>Move the objective to the lowest position.</li>
	<li>Move the objective up until the reflection of the coverslip is visible.</li>
	<li>Increase the detector gain in steps of 50 to 100 V and record the corresponding signal.</li>
	<li>Apply steps 3.1 to 3.8 for both detectors.</li>
	<li>Data evaluation
	<ol>
		<li>Plot the intensity against the detector gain for each detector.</li>
		<li>Choose the individual detector gains to obtain similar sensitivity.</li>
	</ol>
	</li>
</ol>
4. FRET image acquisition
NOTE: Start with the sample of interest for setting up image acquisition.
<ol>
	<li>Choose the appropriate filters/dichroic mirrors, e.g., a double dichroic mirror MBS 458/514 for the FRET-pair ECFP/EYFP. Use the same dichroic mirror for all channels to enable line-by-line scanning. Select a water immersion objective for the imaging of living cells. Choose 12 bit- or 16-bit scanning and moderate scanning speed.</li>
	<li>Define the detection range, preferably 470-510 nm for donor detection and 530-600 nm for acceptor/FRET detection in the case of ECFP/EYFP. When using a 445 nm or 440 nm diode laser, use 450 to 510 nm as the detection range. In the case of an acousto-optic beam splitter (AOBS), define donor detection in the range of 450 to 500 nm to prevent unwanted acceptor detection.</li>
	<li>Apply the detector setting according to 3.10.2.</li>
	<li>Apply the laser setting according to 2.3.2. Revise the laser intensity based on the obtained laser power table, if required. Ensure that the signal-to-noise ratio covers the entire dynamic range of the detectors (intensity ranging from 0 to 4095 for 12-bit scanning).</li>
	<li>Keep laser intensities and detector gains constant. Use the pinhole diameter for fine-tuning. 
	NOTE: Keep in mind that changes in the pinhole diameter affect spatial resolution.</li>
	<li>Perform the measurements (take images of at least 20 cells).</li>
</ol>
5. Determination of crosstalk corrections
NOTE: Cells expressing only the donor or the acceptor are required to determine donor spectral bleed-through (DSBT) and acceptor spectral bleed-through (ASBT), respectively. Keep the same settings described in section 4.
<ol>
	<li>Perform FRET measurements with cells expressing the donor fluorophore.</li>
	<li>Perform FRET measurements with cells expressing the acceptor fluorophore.</li>
</ol>
6. Calibration of the measurements according to Beemiller et al.13
NOTE: Cells expressing a donor-acceptor fusion of known FRET efficiency are required. Here, an ECFP-5 aa-EYFP-fusion with a FRET efficiency of 0.46 has been used4. Keep the same settings described in section 4.
<ol>
	<li>Perform FRET measurements with cells expressing the donor-acceptor fusion</li>
</ol>
7. Data evaluation
<ol>
	<li>Obtain line profiles of the cells, ensuring that each profile contains no more than one cell. Save the profiles as text files.</li>
	<li>Import the text files into a spreadsheet using the text file import option in the Data section.</li>
	<li>Read out the maximum values by applying the Max function.</li>
	<li>List the obtained values in a table, have a column each for donor emission ID, FRET emission IF, acceptor emission IA, and at least four data sets: donor only, acceptor only, donor-acceptor fusion, and measurement. 
	NOTE: Excitation of the donor also results in direct excitation of the acceptor and causes ASBT that is described by the &#945; value.</li>
	<li>Calculate the ASBT &#945; values with the acceptor-only dataset using equation (1). 
	<img alt="Equation 1" src="/files/ftp_upload/62758/62758eq01.jpg" /> (1) 
	NOTE: Use the median of all &#945;-values in the following equations. The donor shows a broad emission spectrum that results in emission crosstalk with the sensitized emission of the acceptor. This DSBT is given by the &#946; value.</li>
	<li>Calculate the donor spectral bleed-through &#946; values with the donor-only dataset using equation (2). 
	<img alt="Equation 2" src="/files/ftp_upload/62758/62758eq02.jpg" /> (2) 
	NOTE: Use the median of all &#946; values in the following equations. The calibration factor &#958; describes the linear relationship of FRET-derived donor quenching and sensitized emission of the acceptor. Use the medians of 7.5 and 7.6 in the following equations.</li>
	<li>Calculate the calibration factors &#958; with the donor-acceptor fusion dataset and its FRET efficiency E (0.46) using equation (3). 
	<img alt="Equation 3" src="/files/ftp_upload/62758/62758eq03.jpg" /> (3) 
	NOTE: Use the median of all &#958; values in the following equations.</li>
	<li>Calculate the FRET efficiencies of the protein pair of interest using equations (4) and (5). 
	<img alt="Equation 4" src="/files/ftp_upload/62758/62758eq04.jpg" /> (4) 
	<img alt="Equation 5" src="/files/ftp_upload/62758/62758eq05.jpg" /> (5)</li>
	<li>Estimate the effects of expression strength and/or donor-acceptor ratio: plot the sum of ID, IF, and IA against the FRET efficiencies. Perform a linear regression; note that the steeper the graph and the higher R2 is, the higher is the impact of the expression level or the greater is the difference of donor and acceptor abundance.</li>
</ol>

<ol>
	<li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescent Spectroscopy. Third Edition. , (2006).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F&#246;rster+resonance+energy+transfer-+FRET+what+it+is,+why+do+it,+and+how+it's+done.">F&#246;rster resonance energy transfer- FRET what it is, why do it, and how it's done.</a> Laboratory Techniques in Biochemistry and Molecular Biology. 33, 1-57 (2009).</li><li>Vogel, S. S., Nguyen, T. A., vander Meer, B. W., Blank, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+heterogeneity+and+dark+acceptor+states+on+FRET:+implications+for+using+fluorescent+protein+donors+and+acceptors.">The impact of heterogeneity and dark acceptor states on FRET: implications for using fluorescent protein donors and acceptors.</a> PLoS ONE. 7, 49593 (2012).</li><li>M&#252;ller, S. M., Galliardt, H., Schneider, J., Barisas, B. G., Seidel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+F&#246;rster+resonance+energy+transfer+by+monitoring+sensitized+emission+in+living+plant+cells.">Quantification of F&#246;rster resonance energy transfer by monitoring sensitized emission in living plant cells.</a> Frontiers in Plant Science. 4, 413 (2013).</li><li>Gadella, T. W. J., vander Krogt, G. N., Bisseling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+FRET-microscopy+in+living+plant+cells.">GFP-based FRET-microscopy in living plant cells.</a> Trends in Plant Science. 4, 287-291 (1999).</li><li>Van Rheenen, J., Langeslag, M., Jalink, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+confocal+acquisition+to+optimize+imaging+of+fluorescence+resonance+energy+transfer+by+sensitized+emission.">Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission.</a> Biophysical Journal. 86, 2517-2529 (2004).</li><li>Seidel, T., Golldack, D., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+C-termini+of+V-ATPase+subunits+by+in+vivo-FRET+measurements.">Mapping of C-termini of V-ATPase subunits by in vivo-FRET measurements.</a> FEBS Letters. 579, 4374-4382 (2005).</li><li>Seidel, T., Schnitzer, D., Golldack, D., Sauer, M., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+iso-enzymes+of+plant+V-ATPase+as+revealed+by+in+vivo-FRET.">Organelle-specific iso-enzymes of plant V-ATPase as revealed by in vivo-FRET.</a> BMC Cell Biology. 9, 28 (2008).</li><li>Schnitzer, D., Seidel, T., Sander, T., Golldack, D., Dietz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+energization+state+affects+peripheral+stalk+stability+of+plant+vacuolar+H+-ATPase+and+impairs+vacuolar+acidification.">The cellular energization state affects peripheral stalk stability of plant vacuolar H+-ATPase and impairs vacuolar acidification.</a> Plant Cell Physiology. 52, 946-956 (2011).</li><li>Roshchina, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+autofluorescence:+application+to+the+study+of+plant+living+cells.">Vital autofluorescence: application to the study of plant living cells.</a> International Journal of Spectroscopy. 2012, 124672 (2012).</li><li>Holtorf, S., Apel, K., Bohlmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+constitutive+and+inducible+promoters+for+the+overexpression+of+transgenes+in+Arabidopsis+thaliana.">Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana.</a> Plant Molecular Biology. 29, 637-646 (1995).</li><li>Seidel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+and+FRET-analysis+of+subunits+c+and+a+of+the+vacuolar+H+-ATPase+in+living+plant+cells.">Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells.</a> Journal of Biotechnology. 112 (1-2), 165-175 (2004).</li><li>Beemiller, P., Hoppe, A. D., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phosphatidylinositol-3-kinase-dependent+signal+transition+regulates+ARF1+and+ARF6+during+FC&#947;+receptor-mediated+phagocytosis.">A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during FC&#947; receptor-mediated phagocytosis.</a> PLoS Biology. 4, 162 (2006).</li><li>Lambert, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FPbase:+a+community-editable+fluorescent+protein+database.">FPbase: a community-editable fluorescent protein database.</a> Nature Methods. 16, 277-278 (2019).</li></ol>

We ensure that all authors have disclosed any and all conflicts of interest and have no competing financial interests.

Donor quenching and sensitized acceptor emission are characterized by a linear relationship that allows for either donor- or acceptor-based calculation of FRET. The corresponding factors of linearity are called either G factor (donor to acceptor) or xi (acceptor to donor), which are reciprocal values4. Measuring FRET between fluorescent proteins by fluorescence microscopy often requires corrections for DSBT and ASBT due to the broad absorption and emission spectra of the fluorescent proteins. However, many corrections depend on a measurable ASBT, which is a factor in the denominator of the equations in protocol section 7, and an &#945; = 0 results in undefined equations5,6.
An adjustment of laser intensities is recommended to avoid such effects. This enables the use of acceptor-based calculations and easy detection of inappropriate donor-acceptor ratios. In this protocol, the equation of Beemiller and co-workers has been applied, which has the advantage of simple calibration by a single reference construct of known FRET efficiency. The adjustment of the detectors is critical, especially when two different types of detectors are applied. Previous data obtained with two identical photomultipliers and identical gains resulted in a constant &#946; = 0.61 for multiple experiments over years7,8,9 and demonstrate that detector adjustment is advantageous for reproducibility. If the detectors do not allow for a reliable adjustment, a sequential scan with a single detector in a frame-by-frame mode might be an option to avoid bias from detector properties.
Measurements in plant cells are affected by many pigments, which result in excitation light absorption and background autofluorescence. The cellular pigments are mainly excited by light in the UV to blue range4,10. This explains the discrepancy between the measurements in cells and those with purified proteins. The purified proteins might serve as a proof of adjustment because the expected values for ASBT &#945; and DSBT &#946; can be derived from the acceptor absorption spectrum and the donor emission spectrum, respectively (Figure 6). The values of ASBT are similar in cells (&#945; = 0.094) and with purified fluorophores (&#945; = 0.114) and close to the expected value of 0.096 due to the low cellular absorption at 514 nm. The DSBT of &#946; = 0.498 with purified proteins is also close to the expected value of 0.406. The spectral bleed-through further depends on the setup; diode lasers of 445 or 440 nm reduce the direct excitation of EYFP and thereby reduce the ASBT. AOBSs are characterized by a smaller transmission gap than dichroic mirrors. Thus, the detection of EYFP is enhanced in the range of 500 to 510 nm and might result in additional ASBT into the donor channel. However, spectral properties of EYFP point to less than 1% emission of the maximum peak, considering both less effective excitation and low emission intensity. The filter gap is much wider with dichroic mirrors, resulting in an additional suppression of such acceptor crosstalk.
Using the values from purified proteins does not reflect the situation in the cell. The same is true for the calibration by the factor &#958;. Although the calibration factor has been similar for both LSM, it was much higher in measurements with the recombinant protein, revealing the impact of cellular pigments. It should be mentioned that in these measurements, the background noise was negligible but detectable and did not affect the observed intensities. In contrast to measurements of the donor lifetime, sensitized emission experiments are sensitive towards the donor-acceptor ratio so that an excess of donor results in decreased FRET efficiency.
However, the protein expression has been under the control of the CaMV35S-promoter in the present experiments. This promoter is frequently used for the overexpression of proteins in plants and might result in unphysiologically high amounts of proteins13. Under such conditions, the probability of interaction increases, and false-positive results might occur with higher protein concentrations. Plotting the emission intensities against the FRET efficiency then results in an increasing FRET efficiency with increasing emission intensity and allows for evaluation of the FRET data with respect to the expression level.
It is recommended to separate high-resolution colocalization experiments from FRET measurements. For colocalization, optimal settings are chosen for each fluorophore, while for FRET recording, emission intensities and fine-tuning by the pinhole diameter interfere with optimal image conditions. Nevertheless, the separation of organelles and information on subcellular structures are still encompassed in FRET measurements.

F&#246;rster resonance energy transfer ((F)RET) is typically observed by fluorescence spectroscopy, although the process itself is not limited to occur between fluorophores. The underlying dipole-dipole coupling simply requires a light-emitting donor molecule and a light-absorbing acceptor. This is derived from the required spectral overlap integral J of the normalized donor emission and acceptor absorbance spectra1. However, because RET competes with fluorescence, the energy transfer becomes measurable by alterations in fluorescence emission: RET induces donor quenching and sensitized acceptor emission.
Fluorophore-based RET has been termed fluorescence resonance energy transfer (FRET) to separate it from bioluminescence resonance energy transfer (BRET). RET depends strongly on the distance between donor and acceptor, which is widely in the range of 0.5-10 nm2 and thus, in the same range as the dimensions of proteins and their complexes. Second, RET depends on the dipole-dipole orientation kappa squared. Combined with the fact that rotational freedom of protein-bound fluorophores can be neglected due to the molecular weight and the slow rotational relaxation, RET allows for the analysis of conformational alterations3.
The so-called F&#246;rster radius is based on the spectral overlap integral and the wavelength range of the overlap, so that red light-absorbing chromophores result in longer F&#246;rster radii than blue light-absorbing dyes. As the dynamic range of FRET measurements is limited by 0.5 &#215; R0 and 1.5 &#215; R0, the FRET pair ECFP-EYFP has a dynamic range of 2.5-7.3 nm due to its R0 of 4.9 nm4.
The brightness of a fluorophore is given by the product of its molar extinction coefficient and its quantum yield. For FRET measurements, it is advantageous to choose fluorophores of nearly similar brightness. This enhances the detection of donor quenching and sensitized acceptor emission. It also favors the calibration of the microscopy system. Looking at the frequently used FRET pairs of cyan and fluorescent proteins, the lower brightness of the cyan fluorescent proteins becomes obvious (Figure 1A).
However, the lifetime of the acceptor must be lower than the lifetime of the donor, ensuring the availability of the acceptor for energy transfer. If the lifetime of the acceptor exceeds the lifetime of the donor, the acceptor might still be in the excited state when the donor is excited again. Advanced cyan fluorescent proteins such as mTurquoise show an extended lifetime and thus contribute to an increased probability of FRET (Figure1B). The probability of FRET also depends on the molar extinction coefficient of the acceptor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well slides</td>
			<td>Ibidi</td>
			<td>80821</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil Immersol&#160; W2010</td>
			<td>Zeiss</td>
			<td>444969-0000-000</td>
			<td>refraction index of water</td>
		</tr>
		<tr>
			<td>LSM 1: AxioObserver with LSM 780 scan head, confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 2: AxioObserver with LSM 5 scan head, confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

f&#246;rster resonance energy transfer measurements in living plant cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

Adjustment of the confocal laser-scanning microscope 
The laser adjustment revealed a linear increase of emission with increasing laser intensity (Figure 2 and Table 1). As expected for argon-ion lasers, the emission of the 514 nm line was much higher than the emission of the 458 nm line, as evidenced by a steeper slope. For subsequent experiments, laser power of 4.5% and 6.5% was chosen for the 514 nm line and the 458 nm line, respectively. This resulted in almost equal emission intensity of 1123 (514 nm) and 1141 (458 nm).
Varying the detector gains at constant laser power revealed an exponential behavior for both analyzed detectors. Similar emission intensities were obtained for a gain of 700 V (Figure 3). Although the adjustment of laser lines benefited from the linear behavior, the adjustment of the detectors is likely affected by the exponential behavior, resulting in marked differences with minor changes in the gain at higher voltages. Unfortunately, this higher range is of interest for measurements in living cells, as increasing the laser power is cytotoxic and promotes photobleaching.
Determining spectral bleed-through 
At first, the spectral bleed-through of the donor and the acceptor were analyzed with recombinant purified ECFP and EYFP; DSBT &#946; = 0.498 and ASBT &#945; = 0.100. The same was done with cells expressing either ECFP or EYFP. With LSM 2, the estimated DSBT was &#946; = 1.602 &#177; 0.207 (mean &#177; SD) and ASBT was &#945; = 0.119 &#177; 0.018. The median values were &#946; = 1.506 and &#945; = 0.120. This discrepancy between the data obtained in living cells and the data obtained with recombinant protein demonstrates that it is impossible to omit the determination of spectral bleed-through in living cells. This is likely caused by cellular pigments.
The images reveal the higher brightness of EYFP in comparison to ECFP (Figure 4 and Table 2). Compensating the differences in brightness by different laser settings enhances the dynamic range of the donor and the FRET channel. Providing the relative output by measuring the laser intensities, as done for the adjustment, still increases the reproducibility of the measurements.
For LSM 1, &#946; = 0.171 &#177; 0.044, &#945; = 0.094 &#177; 0.031 with medians of &#945; = 0.084 and &#946; = 0.180. The determination of the ASBT depends on the laser adjustment and a single detector, and thus, the determination of ASBT performed well. The DBST involves two detectors, resulting in vastly different results for both microscopes. It should be remembered that LSM 2 is equipped with two photomultipliers, whereas LSM 1 uses a GaAsP detector and a photomultiplier, two different types of detectors with different spectral properties and sensitivity. Accordingly, the adjustment works better with two identical detectors.
Calibration of the measurement 
For calibration, the median values of &#945; and &#946; were applied, and a fusion of ECFP and EYFP was used as the standard of known FRET efficiency (E = 0.46). The calibration of recombinant and purified ECFP-EYFP resulted in a &#958; value of 13.44, while the in vivo measurements revealed a &#958; value of 1.525 &#177; 1.844. The median was 0.798, revealing extreme outliers together with the high standard deviation. For LSM 1, the values were &#958; = 1.978 &#177; 0.807 with a median of &#958; = 1.883. For LSM 2 and LSM 1, the datasets obtained for the calculation of &#958; (Table 3) were statistically identical, as proven by Student&#39;s t-test (p &#62; 0.2).
FRET measurement 
As a proof of concept, the FRET measurement was repeated with the donor-acceptor fusion. The measured FRET efficiency was 0.47 &#177; 0.07 for the purified protein and 0.47 &#177; 0.06 in living cells with LSM 2 (Table 4). The median of the measurements in living cells was E = 0.45. For LSM 1, the FRET efficiency was 0.46 &#177; 0.09 with a median of E = 0.45 (Table 4). These data demonstrate the effective calibration with a FRET construct of known FRET efficiency.
Effects of expression level and donor-acceptor ratio 
For the analyzed data set, the FRET efficiencies were not influenced by the expression level. Due to the fusion of the donor and the acceptor, the ratio was also constant. The inclusion of previous datasets revealed a dependency on the expression level in one case (Figure 5). The FRET efficiency between the labeled vacuolar ATPase (V-ATPase) subunits, VHA-A-ECFP and VHA-a-EYFP, decreased with increasing signal intensity. In contrast, the interaction between VHA-E1-ECFP and VHA-C-EYFP was independent of the signal intensity. In the case of VHA-A and VHA-a, the three copies of VHA-A are increasingly replaced by VHA-A-ECFP, which results in a donor excess in comparison to the single copy of VHA-a in the complex. Although VHA-E1 might be present in the form of three copies, the high solubility of VHA-C might abolish this effect in this example. Here, the FRET efficiencies have been comparatively low. This simple approach allows for the testing of expression and ratio artifacts.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62758/62758fig01.jpg" /> 
Figure 1:&#160;Overview of fluorescent protein FRET pairs with cyan-emitting donors. (A) The F&#246;rster radii of the pairs and the brightness of donor and acceptor are given for well-characterized FRET pairs. (B) Comparison of donor and acceptor lifetimes. Grey asterisks indicate a multiexponential decay. Abbreviations: FRET = F&#246;rster resonance energy transfer; CFP = cyan fluorescent protein; GFP = green fluorescent protein; YFP = yellow fluorescent protein; mX = monomeric X; EX = enhanced X; SX = super X. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/62758fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62758/62758fig02.jpg" /> 
Figure 2:&#160;Laser adjustment. The emission of the lasers was recorded by the reflection of the coverslip and plotted against the relative laser intensity as modulated by the acousto-optic tunable filter of the LSM. Emission of the 458 nm line (A) and the 514 nm line (B) of an argon-ion laser is shown. Abbreviations: LSM = laser scanning microscope; r.u. = relative units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/62758fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62758/62758fig03.jpg" /> 
Figure 3:&#160;Detector gain and emission intensity. The resulting emission intensities were recorded in the reflection mode for detector gains ranging from 300 to 750 V and 300 to 750 V for detector 1 (A) and detector 2 (B), respectively. Abbreviation = r.u. = relative&#160;units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/62758fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62758/62758fig04.jpg" /> 
Figure 4:&#160;Spectral bleed-through. Images were obtained in the donor, the FRET, and the acceptor channels. (A) Images obtained with purified fluorescent proteins; scale bars = 100 &#181;m; (B) corresponding images for cells expressing the fluorescent proteins; scale bars = 10 &#181;m. (C) Graphs of the obtained SBT values; mean &#177; SD is given. Abbreviations: FRET = F&#246;rster resonance energy transfer; SBT = spectral bleed-through; ECFP = enhanced cyan fluorescent protein; EYFP = enhanced yellow fluorescent protein; LSM = laser scanning microscope; ASBT = acceptor SBT; DSBT = donor SBT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/62758fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62758/62758fig05.jpg" /> 
Figure 5:&#160;Effects of expression level and/or donor-acceptor ratio. The sum of the emissions in the donor, FRET, and acceptor channels has been plotted against the FRET efficiency. This has been done for VHA-E1-ECFP and VHA-C-EYFP (A), VHA-A-ECFP and VHA-a-EYFP (B), and the ECFP-EYFP fusion (C). Linear regression was performed; the equation and R2 are given. Abbreviations: FRET = F&#246;rster resonance energy transfer; ECFP = enhanced cyan fluorescent protein; EYFP = enhanced yellow fluorescent protein; VHA = vacuolar ATPase subunit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/62758fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62758/62758fig06.jpg" /> 
Figure 6:&#160;Donor and acceptor bleed-through of ECFP and EYFP. The spectra of the fluorescent proteins were obtained from the FP database14. (A) During sensitized emission experiments, the vast portions of the donor and the acceptor are detected by individual photomultipliers, named PMT1 and PMT2, respectively. ECFP emission is also detectable by the acceptor detector upon excitation with 458 nm. The calculated donor spectral bleed-through into PMT2 is 40.6% of the emission detected by PMT1. (B) The emission spectrum of EYFP demonstrates that EYFP shows direct excitation at 458 nm, which is frequently applied for the excitation of cyan donors. The expected excitation efficiency is 9.6% of the excitation at 514 nm. Abbreviations: FP = fluorescent protein; ECFP = enhanced cyan fluorescent protein; EYFP = enhanced yellow fluorescent protein; PMT = photomultiplier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/62758fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1:&#160;Laser power and signal intensity. The signal intensities of the 458 nm line are given in blue, the signal intensities of the 514 nm line in green. The intensities applied for the experiment are highlighted in grey. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2:&#160;Signal intensities for SBT determination. Recorded maxima and calculated &#945; and &#946; values are given. Abbreviations: SBT = spectral bleed-through; LSM = laser scanning microscope; FRET = F&#246;rster resonance energy transfer; ASBT = acceptor SBT; DSBT = donor SBT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3:&#160;Signal intensities for calibration. Recorded maxima and calculated &#958; values are given. Corrected values correspond to the FRET signal intensities minus SBT. Abbreviations: SBT = spectral bleed-through; LSM = laser scanning microscope; FRET = F&#246;rster resonance energy transfer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4:&#160;Signal intensities for FRET measurements. Recorded maxima and E values are given. Corrected values correspond to the FRET signal intensities minus SBT. Calibrated E values were calculated with calibration by &#958;. Abbreviations: SBT = spectral bleed-through; LSM = laser scanning microscope; FRET = F&#246;rster resonance energy transfer; cal. = calibrated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62758/Table 4.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about FÃƒÂ¶rster Resonance Energy Transfer Measurements in Living Plant Cells at JoVE.com

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

frster-resonance-energy-transfer-measurements-in-living-plant-cells

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.7K Views.  Bielefeld University. A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo Förster resonance energy transfer measurements, followed by data evaluation.

Video: Förster Resonance Energy Transfer Measurements in Living Plant Cells

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

We present protocols herein for high-yield isolation of physiologically active thylakoids and protein transport assays for the chloroplast twin arginine translocation (cpTat), secretory (cpSec1), and signal recognition particle (cpSRP) pathways.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

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 ...

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.
Metabolic profiling by 1H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop&#160;conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical 1H NMR profiling.
In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to 1H NMR classical spectra.
Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant&#160;leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...