Name: spin saturation transfer difference nmr (sstd nmr): a new tool to obtain kinetic parameters of chemical exchange processes
Uploaded: 2016-11-12T13:00:00.000+00:00
Duration: 11 min 44 s
Description: Watch this Scientific Journal Video about Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes at JoVE.com

Funding by the University of East Anglia, the EPSRC (EP/L012855/1) and the EU (H2020-MSCA-IF-2014-EF-ST-658172) is gratefully acknowledged (MTQ).

Caution: Please consult all relevant material safety data sheets (MSDS) before use.

1. NMR Sample Preparation
<ol>
	<li>Weigh 5 mg of N,N-dimethylacetamide, add to an NMR tube appropriate for low temperatures and dissolve in 0.6 ml of toluene-d8.</li>
</ol>
2. NMR Experimental Setup25
<ol>
	<li>NOE Spectra Acquisition
	<ol>
		<li>Perform a one dimensional NOE (Nuclear Overhauser Effect) experiment.26 
		NOTE: NOE effects can happen at any temperature. A one dimensional NOE spectrum irradiating the signal that will be irradiated in the SSTD NMR experiment, was recorded at -40 &#176;C to make sure that the rotation and magnetization transfer in the sample used here was minimized, and therefore the NOE, if existent, would predominate and be measured in this experiment. Ideally, NOE effects between the two exchanging nuclei should not be present to avoid interferences with the SSTD method.</li>
	</ol>
	</li>
	<li>SSTD NMR Experiments Setup
	<ol>
		<li>Insert the sample in the magnet by first typing ej in the command line of the software to turn on the air flow. Then, put the sample on top of the magnet and then type ij. Wait until the sample is inside the magnet.</li>
		<li>Once the sample is in the magnet, type edte in the command line. Change the temperature to the first selected temperature to carry out the experiment (295.5 K in this case). Let the sample stabilize at the chosen temperature for at least 20 min.</li>
		<li>Perform a 1D 1H-NMR experiment on the sample.
		<ol>
			<li>Create a new dataset of a 1H-NMR experiment. For this click on FILE/NEW and name the new experiment.</li>
			<li>Type sequentially and waiting for the previous command to finish: lock, atma, topshim, getprosol and rga.</li>
			<li>Type zg to acquire the proton experiment. Once it is finished type efp and apk to Fourier transform it and adjust the phase.</li>
		</ol>
		</li>
		<li>Create a new dataset of, for example, a 1H NMR experiment. For this click on FILE/NEW and name the new experiment.</li>
		<li>In this new dataset, type rpar in the command line. Select one of the &#34;STDDIFF&#34; parameter sets from the list, for example STDDIFFESGP, and click &#34;read&#34; and then &#34;read all&#34; (Figure 1). Alternatively, do this by typing rpar STDDIFFESGP all. 
		NOTE: The experiment can be performed with this pulse sequence. However, the pulse program used in our experiment was STDDIFF.</li>
		<li>To select the STDDIFF pulse sequence, click the button with three dots in the PULPROG line (Figures 2 and 3).</li>
		<li>Before carrying out the SSTD NMR experiment, calibrate the 1H 90&#176; hard pulse (p1). For this purpose, ensure that the sample is in the magnet at the desired temperature (Step 2.2.2). Type pulsecal in the command line and copy the value of the 90&#176; pulse at the higher power (pl1 = -1 dB in this case), i.e., the one that gives the shortest pulse (Figure 4).</li>
		<li>Introduce the values for the calibrated hard pulse in the experiment. Type getprosol 1H (value for p1 obtained in step 2.2.7) (value for pl1) (Figure 5).</li>
		<li>Set the length of the shaped pulse. Type p13 and introduce a value of 50,000 &#181;sec (Figure 6).</li>
		<li>Set the selective pulse shape. To do this, go to Power and click the &#34;Edit&#8230;&#34; button next to SHAPE (Figure 7). Go to the shaped pulse 13 and choose: Gaus 1.1000 (Figure 8).</li>
		<li>Set the selective pulse power (SP13). Set it to something appropriate, i.e., on this system between 40-60 dB (corresponding to a field strength of approximately 120 Hz) (Figure 8). Excessive field strengths can lead to unacceptable saturation effects.27-29 
		NOTE: 50 dB was optimum in our case. Take into account that this is an attenuation scale, so the smaller the value the higher the power of the radiofrequency. As it corresponds to the saturating Gaussian cascade, which is applied for long time (several seconds), sp13 should not go below 40 dB (if needed, consult the instrument specifications, as long pulses at high power could damage the probehead). In our experience 41-61 dB above the attenuation of the hard 1H 90&#176; pulse (-1 dB in this work) works fine. Try to always select the highest attenuation possible leading to similar saturation level.</li>
		<li>Type ns and set it to 8 and type ds and set it to 4.</li>
	</ol>
	</li>
</ol>
3. NMR Data Acquisition and Processing25
<ol>
	<li>SSTD NMR Experiment Acquisition
	<ol>
		<li>Open the 1H NMR experiment performed in step 2.2.3 to check where the signal that will be irradiated is. For this, search the experiment in the software browser, right click in the dataset and click &#34;Display in a new window&#34;.</li>
		<li>Move the cursor line to the center of the signal to irradiate and write down the chemical shift in ppm. Select the spectral width that will be used in the experiment. 
		NOTE: In this case, the signal which will be irradiated is at 2.17 ppm, and the spectral width used was 1.46 ppm. Ensure that no chemical shift correction is used or the irradiation frequency can be set incorrectly.</li>
		<li>Go to the previously created SSTD NMR experiment with the setup mentioned in section 2.2.</li>
		<li>Create a list with the frequencies of irradiation. For this, type fq2list in the command line and select an existing list.</li>
		<li>Edit the list of irradiation frequencies including the following data in the 3 first rows (Figure 9): Row 1. P (indicates that the following data is in ppm); Row 2. Frequency of the signal to be irradiated in ppm, as measured in 3.1.1; Row 3.40 ppm (a frequency that is far from the 1H signals of the compound so the irradiation in that frequency does not affect the spectra).</li>
		<li>Save the list with a new name and then type fq2list in the command line and select the list just created.</li>
		<li>To center the experiment on the signals under study, type o1p and select as the center of the experiment the chemical shift of the signal that will be irradiated.</li>
		<li>Type sw to select the spectral width (1.46 ppm in this case, but any other spectral width can be chosen). 
		NOTE: If the acquisition time obtained after changing the spectral width is too long (which will introduce more noise in the spectra) it can be adjusted by typing AQ to provide the desired Free Induction Decay (FID) resolution (FIDRES, 0.25 Hz in this case).</li>
		<li>Choose the value for the interscan relaxation delay D1. Ensure that it is at least 1 to 5 times the value of the T1 of the slowest relaxing proton. 
		NOTE: We set it up to 40 sec, which is the longest saturation time (D20) in the experiment. In this way all of the experiments will keep the same total &#34;per scan&#34; time (delay + saturation time + pulses + acquisition time).</li>
		<li>Type D1 and set it to 40 sec.</li>
		<li>Set the first value for the saturation time by typing D20 and setting it to 40 sec. Determine the receiver gain (rg) automatically by typing rga.</li>
		<li>Create the next experiment by typing iexpno. Type D20 and select a saturation time of 20 sec in this experiment. Type rga to automatically determine rg.</li>
		<li>Repeat the last step for D20 = 10, 5, 2.5, 1.25, 0.625, 0.3 sec.</li>
		<li>Once all the experiments are created, open the first one and in the command line type multizg and specify the number of experiments, 8 in this case (i.e., multizg 8).</li>
	</ol>
	</li>
	<li>SSTD NMR Experiment Processing
	<ol>
		<li>Open the PROCNO 1 (Process Number) from EXPNO 1 (Experiment Number) of the set (the one with the higher saturation time).</li>
		<li>In the command line type lb and set the value to 1.5. 
		NOTE: For spectra with very high signal-to-noise ratios this value could be decreased; inversely, it could be increased for noisy experiments, if the spectral resolution is not severely affected.</li>
		<li>In the command line type efp and process FID # = 1 (&#34;on-resonance&#34; spectrum) in PROCNO = 2 (Figure 10).</li>
		<li>Correct the phase of the experiment by clicking the Interactive phase correction button and save it as a 2D experiment. Save and exit (Figure 11).</li>
		<li>Type rep 1 in the command line to go to the PROCNO 1.</li>
		<li>In the command line type efp and process FID # = 2 (&#34;off-resonance&#34; spectrum) in PROCNO = 3 (Figure 12).</li>
		<li>In the command line type .md and then rep 2 to show a multiple display window with both processed spectra: 2 (the one with the signal in the middle saturated) and 3 (the one in which the saturating pulse train was applied at 40 ppm) (Figure 13).</li>
		<li>Click the button with a delta sign (Figure 13) to calculate the difference spectra and save it in PROCNO 4. Exit the multiple display window.</li>
		<li>Select an integration range for the signal on the left (the signal in which the transfer of saturation due to the chemical exchange process will be observed). Always integrate the same region in PROCNO 3 and PROCNO 4. 
		NOTE: The integration range used in this experiment was 2.55 - 2.67 ppm.</li>
		<li>Once integrated, go to the &#34;Integrals&#34; tab in each of the experiments and copy the value of &#34;Integral [abs]&#34; (Figure 14).</li>
		<li>Divide the integral in PROCNO 4 by the integral in PROCNO 3. That is the value of &#951;SSTD for a saturation time of 40 sec (&#951;SSTD =Spin Saturation Transfer Difference parameter).21</li>
		<li>Repeat the procedure for the rest of the experiments with different saturation times.</li>
	</ol>
	</li>
</ol>
4. Data Analysis30
<ol>
	<li>Analysis of the Data to Get the Kinetic Parameters
	<ol>
		<li>Plot the obtained &#951;SSTD values versus the saturation time.21</li>
		<li>Perform an exponential fit to adjust the obtained curves to the equation 
		<img alt="Equation 1" src="/files/ftp_upload/54499/54499eq1.jpg" /> 
		<img alt="Equation 2" src="/files/ftp_upload/54499/54499eq2.jpg" /> 
		<img alt="Equation 3" src="/files/ftp_upload/54499/54499eq3.jpg" />=&#160;<img alt="Equation 4" src="/files/ftp_upload/54499/54499eq4.jpg" /> at very long saturation time 
		t = time</li>
		<li>Calculate the values of &#951;SSTDMAX and &#948; and use them to calculate the values of the rate constants (k) and relaxation times (T1A) according to the following equations: 
		<img alt="Equation 5" src="/files/ftp_upload/54499/54499eq5.jpg" /> 
		T1A = longitudinal relaxation time constant of spin A 
		k = mutual-site exchange kinetic rate constant
		<ol>
			<li>Obtain the kinetic rate constant by: <img alt="Equation 6" src="/files/ftp_upload/54499/54499eq6.jpg" /></li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Eyring Plot to get the Thermodynamic Parameters
	<ol>
		<li>Plot ln(k/T) versus 1/T (T = absolute temperature), using the values of the kinetic rates at different temperatures.</li>
		<li>Perform a linear fit to adjust the data obtained to the Eyring equation: 
		<img alt="Equation 7" src="/files/ftp_upload/54499/54499eq7.jpg" /> 
		<img alt="Equation 8" src="/files/ftp_upload/54499/54499eq8.jpg" /> 
		<img alt="Equation 9" src="/files/ftp_upload/54499/54499eq9.jpg" /> 
		<img alt="Equation 10" src="/files/ftp_upload/54499/54499eq10.jpg" /> 
		R = gas constant 
		kB = Boltzmann constant 
		T = absolute temperature</li>
		<li>Calculate the thermodynamic parameters &#916;H&#8800; and &#916;S&#8800;.</li>
		<li>Calculate the values for EA(298) and &#916;G&#8800;(298) using the following equations: 
		<img alt="Equation 11" src="/files/ftp_upload/54499/54499eq11.jpg" /> 
		<img alt="Equation 12" src="/files/ftp_upload/54499/54499eq12.jpg" /> 
		<img alt="Equation 13" src="/files/ftp_upload/54499/54499eq13.jpg" /> 
		<img alt="Equation 14" src="/files/ftp_upload/54499/54499eq14.jpg" /> 
		&#160;</li>
	</ol>
	</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+Internal+Rotation+of+N,N-Dimethylacetamide:+A+Spin-Saturation+Transfer+Experiment.">Kinetics of Internal Rotation of N,N-Dimethylacetamide: A Spin-Saturation Transfer Experiment.</a> J. Chem. Ed. 74, 978-982 (1997).</li><li>Forsen, S., Hoffman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+Rates+by+Nuclear+Magnetic+Multiple+Resonance.+III.+Exchange+Reactions+in+Systems+with+Several+Nonequivalent+Sites.">Exchange Rates by Nuclear Magnetic Multiple Resonance. III. Exchange Reactions in Systems with Several Nonequivalent Sites.</a> J. Chem. Phys. 40, 1189-1196 (1964).</li></ol>

The authors declare that they have no competing financial interests.

One of the more obvious advantages of this methodology is that the rate constants and the relaxation time for a given temperature can be obtained with a single set of experiments, with a robust pulse sequence (the same used for STD experiments to study protein-ligand interactions, which is typically found within the available set of experiments from the spectrometer manufacturer). This simplifies the experimental setup since there is no need to measure T1 or reach steady state saturation. Besides, it is remarkable that this method does not depend on the magnet strength, as coalescence methods. On the other hand, the main limitation is that this technique cannot be applied to chemical exchange processes too fast or too slow, which would depend on the temperature range of the NMR machine or the solvents used.
This new technique for the calculation of kinetic parameters can be applied to a great variety of substrates and its applicability has already been demonstrated with some interesting molecules.21 The kinetic parameters of the 4-N,N-dimethylamido[2.2]paracyclophane, a challenging substrate in which the signal of one of the methyl groups of interest is overlapped with other signals from the molecule, were successfully calculated using SSTD NMR. Interestingly, this methodology can be applied as long as one of the signals of study is isolated. SSTD NMR is also a useful protocol for the calculation of kinetic parameters in molecules in which the coalescence temperature is so high that the molecule decomposes before reaching it. This is the case with PtCl2(dimethylallene)(pyridine), in which the methodology was successfully applied without the need of reaching coalescence. The choice of solvents and temperatures is critical to obtain good results, since the chemical exchange rates can vary significantly with these parameters. Moreover, in addition to the criteria in a normal NMR experiment, key steps in a SSTD NMR experiment are the selectivity of the irradiation as well as the temperature control. Both factors have to be precise to guarantee the success of the experiment.
The representative results presented here are for the kinetics of intramolecular chemical exchange, but the technique can also be applied to study the kinetics of intermolecular chemical exchange and also ligand exchange, common processes in the dynamic behavior of transition metal complexes. 
Finally, providing a proper modification of the equations is made,32 this method could be extended to deal with multi-site exchange and unequal populations, as it has been done in former double resonance experiments,8-9 increasing the usefulness of this technique for the study of chemical exchange processes in challenging compounds.

Chemical exchange commonly refers to any intermolecular or intramolecular process in which a nucleus moves from one environment to another in which its NMR parameters (chemical shift, scalar coupling, dipolar coupling, relaxation rate) differ. There are numerous examples of chemical exchange in organic and organometallic molecules (e.g., rotational barriers in biaryls, ring flipping barriers and conformational equilibrium, nitrogen inversion, ligand binding, degenerate ligand exchange and tautomerization).1-3 The chemical exchange rate is related to the thermodynamics of the barrier of the exchange process, and therefore its study is of crucial importance to understand molecular dynamics of these systems.
The classic sign of dynamic exchange in NMR is a dramatic change in the line-shape of the NMR signals as the temperature changes. At low temperatures, the process is slow and two distinct chemical shifts are observed. At high temperatures, the two signals merge into one signal, which is known as &#34;coalescence&#34;. At intermediate temperatures, the signals become very broad. This sensitivity of the NMR spectrum to chemical exchange makes NMR a very powerful method to study the dynamics of molecules in solution. Two methods have been mainly employed in the study of dynamic processes in solution: line-shape analysis,4-7 and spin saturation transfer experiments.8-9 Besides, it is also worth mentioning the inversion transfer method10 and the CIFIT program11 for the direct extraction of rate constants, that are a relatively efficient approach for exchange measurements in simple systems. Although these methods give very good results in most cases, they, however, have a number of drawbacks. The main disadvantage of the line-shape analysis is the high temperatures needed to reach coalescence in some samples.12 The main issues to consider when carrying out spin saturation transfer experiments are: the very long saturation times required to reach the steady state saturation transfer between the exchanging sites, and the need to determine the longitudinal relaxation time constant, T1, which can be difficult if there is overlap of different signals in the region of study.13
As part of our investigations in organometallic mechanisms,14-16 our group is studying the fluxional behavior of platinum-allene complexes in solution. This is a complex task that involves at least three different processes, one of them being the &#960;-face exchange or rotation of the metal around one of the allene axis. We encountered that normal VT experiments and line-shape analysis techniques that have been employed before in similar systems,17-19 were not suitable in our study, due to a very slow rotation in our platinum-allene complex that made the coalescence temperature of the signals of interest higher than the temperature of decomposition of the complex.
In order to overcome this limitation, we developed and recently reported a new NMR protocol (SSTD NMR) to study processes of mutual-site chemical exchange.20 As the name suggests this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference NMR method employed for the study of protein-ligand interactions,21-24 by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small molecules undergoing chemical exchange.
With this new method (SSTD NMR) we have shown that we can obtain the kinetic parameters of intramolecular chemical exchange in small organic and organometallic molecules with some additional advantages over traditional approaches: coalescence of the signals is not needed, so a more flexible temperature range can be used in the study; signal overlap does not interfere, although at least one of the exchanging resonances should be isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly and T1 values are obtained in the same experiment, using only one set of experiments. Another remarkable advantage of the SSTD NMR methodology is that, in contrast to lineshape analysis, the determination of the kinetics rate constants is not limited by the increase in coalescence temperatures associated with high magnetic fields. Thus, our methodology is then very well appropriated for both low and high magnetic fields. This article is intended to help new users apply this new method to their own systems undergoing chemical exchange, and describes sample preparation, experimental set up, data acquisition, and an example of data processing and analysis in a simple organic molecule.

<table><tbody><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethylacetamide</td><td>Aldrich</td><td>38840</td><td>Acute toxicity</td></tr><tr><td>Toluene-&lt;em&gt;d&lt;sub&gt;8&lt;/sub&gt;&lt;/em&gt;</td><td>Fluorochem</td><td>D-005</td><td>Flammable and toxic</td></tr><tr><td>500 MHz 7&quot; Select Series NMR Tubes</td><td>GPE LTD</td><td>S-5-500-7</td><td></td></tr><tr><td>TopSpin 2.1</td><td>TopSpin program, Bruker Corp., http://www.bruker.com/products/mr/nmr/nmr-software/software/topspin/ (2015).</td><td></td><td></td></tr><tr><td>Origin 6.0</td><td>Origin 6.0 software, OriginLab Corp., http://originlab.com.</td><td></td><td></td></tr><tr><td>Bruker Avance III 500 MHz fitted with 5 mm broadband observed BBFOplus Z-gradient SmartProbeTM probe</td><td>&amp;nbsp;Bruker Corp., http://www.bruker.com</td><td></td><td></td></tr><tr><td>Bruker Avance I 500 MHz Inverse Triple Resonance NMR spectrometer fitted with a 5 mm TXI Z-gradient probe</td><td>&amp;nbsp;Bruker Corp., http://www.bruker.com</td><td></td><td></td></tr><tr><td>Ceramic Spinner standardbore shimsystems (5 mm)</td><td>&amp;nbsp;Bruker Corp., http://www.bruker.com</td><td>H00804</td><td></td></tr></tbody></table>

spin saturation transfer difference nmr (sstd nmr): a new tool to obtain kinetic parameters of chemical exchange processes

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our group to study processes of mutual-site chemical exchange that are difficult to analyze by traditional methods. As the name suggests, this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference (STD) NMR method employed for the study of protein-ligand interactions, by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small organic and organometallic molecules undergoing chemical exchange.
Advantages of this method over existing ones are: there is no need to reach coalescence of the exchanging signals; the method can be applied as long as one signal of the exchanging sites is isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly, and T1 values are obtained in the same experiment, using only one set of experiments.
To test the method, we have studied the dynamics of the hindered rotation of N,N-dimethylamides, for which much data is available for comparison. The thermodynamic parameters obtained using SSTD are very similar to the reported ones (spin-saturation transfer techniques and line-shape analysis). The method can be applied to more challenging substrates that cannot be studied by previous methods.
We envisage that the simple experimental set up and the wide applicability of the method to a great variety of substrates will make this a common technique amongst organic and organometallic chemists without extensive expertise in NMR.

The SSTD NMR technique was applied for the calculation of the kinetic parameters in the rotation of the amide bond of N,N-dimethylacetamide.21 This is a simple example for which extensive data for comparison can be found in the literature.31
The hindered rotation around the amide bond, due to the partial double bond character in the resonance form, differentiates both methyl groups into two signals in the 1H-NMR spectra (2.61 and 2.17 ppm at 22.5 &#176;C). Spin saturation of the signal of the methyl group at 2.17 ppm (MeB) leads to the disappearance of its signal in the 1H NMR. Upon saturation of MeB, transfer of saturation to the other methyl group (MeA) due to the internal rotation process can be observed by a decrease in 1H intensity in the signal at 2.61 ppm. The magnitude of this decrease will depend on the saturation time. Figure 15 shows the 1H NMR spectra of the N,N-dimethylacetamide at 22.5 &#176;C, and the expansions show the spectra without (a) and with (b) saturation of the methyl group at 2.17 ppm, as well as the difference spectrum (c), used to calculate the values of &#951;SSTD. The &#951;SSTD factor is calculated dividing the value of the integral of MeA in the SSTD NMR spectrum (c) by the value of the integral of the MeA in spectra (a), as explained in the protocol. The obtained values of &#951;SSTD for each saturation time at different temperatures are gathered in Table 1. The plot of the obtained values of &#951;SSTD versus the saturation time gave exponential curves in which a plateau was reached at higher saturation times. For a certain temperature, the exponential fit of the curve permits the calculation of the rate constant (k) and the relaxation time of the 1H of the measured signal (T1A) (Figure 16). Figure 17 shows all the obtained curves along with the k and T1A values obtained in the fits.
Finally, the plot of ln(k/T) versus 1/T and the fit to the Eyring equation (Figure 18) were used to calculate the enthalpy and entropy of activation. The determined activation parameters are shown in Table 2, together with the previously reported parameters calculated using different methodologies.
As can be observed in Table 2, the values of the activation parameters obtained with the Spin Saturation Transfer Difference technique (SSTD NMR) are in excellent agreement with the data previously reported using other techniques, such as SST NMR or line shape analysis. The wide range of values reported for &#916;S&#8225; is due to the difficulty in the measurement of this parameter with NMR techniques.31 As for the rest of the activation parameters, the values obtained with our method are not only really similar to the ones already reported but also more accurate, since our errors (SD) are smaller in all the cases.
<img alt="Figure 1" src="/files/ftp_upload/54499/54499fig1.jpg" /> 
Figure 1: List of experiments after typing rpar. The figure shows the different parameter sets among which STDDIFFESGP should be selected. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54499/54499fig2.jpg" /> 
Figure 2: Acquisition parameters. The button highlighted in a red square leads to a list of the different pulse programs. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54499/54499fig3.jpg" /> 
Figure 3: List of pulse programs. The figure shows the selected pulse program in the experiment (STDDIFF). <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54499/54499fig4.jpg" /> 
Figure 4: Pop-up window appeared after the 90&#176; pulse calibration. The figure shows the values of the calibrated 90&#176; pulse at different power levels. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54499/54499fig5.jpg" /> 
Figure 5: Screenshot of the command line. The figure shows how to introduce the value for the calibrated hard pulse. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54499/54499fig6.jpg" /> 
Figure 6: Value for the length of the shaped pulse. The figure shows how to introduce the value for the length of the shaped pulse. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54499/54499fig7.jpg" /> 
Figure 7: Acquisition parameters. The figure shows the power parameters. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54499/54499fig8.jpg" /> 
Figure 8: Parameters for the shaped pulse. The values for the shaped pulse will be introduced in line 13. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54499/54499fig9.jpg" /> 
Figure 9: List of irradiation frequencies. The figure includes the following data in the 3 first rows: Row 1. P (indicates that the following data is in ppm); Row 2. Frequency of the signal to be irradiated in ppm, as measured in 3.1.1; Row 3.40 ppm (a frequency that is far from the 1H signals of the compound so the irradiation in that frequency does not affect the spectra). <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/54499/54499fig10.jpg" /> 
Figure 10: Processing of the first FID. The figure shows the pop up window that appears after typing efp. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" src="/files/ftp_upload/54499/54499fig11.jpg" /> 
Figure 11: Phase correction. Screenshot showing the window for the manual phase correction. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" src="/files/ftp_upload/54499/54499fig12.jpg" /> 
Figure 12: Processing of the second FID. The figure shows the pop up window that appears after typing efp. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" src="/files/ftp_upload/54499/54499fig13.jpg" /> 
Figure 13: Multiple display of spectra 2 and 3. The button highlighted in a red square is the one to calculate the difference spectra. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" src="/files/ftp_upload/54499/54499fig14.jpg" /> 
Figure 14: Integrals tab. The figure shows the values of the absolute and relative integrals. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" src="/files/ftp_upload/54499/54499fig15.jpg" /> 
Figure 15: Structure and 1H NMR spectra of N,N-dimethylacetamide at 22.5 &#176;C in toluene-d8. (a) 1H NMR expansion of the region from 2.13 to 2.66 ppm before irradiation. (b) Expansion of the same region after irradiation of the methyl group at 2.17 ppm. (c) Difference spectrum [(a)-(b)]. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 16" src="/files/ftp_upload/54499/54499fig16.jpg" /> 
Figure 16: Example of the plot of &#951;SSTD and its exponential fit at 278 K. Reproduced from the supporting information of reference21 with permission from the Royal Society of Chemistry. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig16large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 17" src="/files/ftp_upload/54499/54499fig17.jpg" /> 
Figure 17: Plots of &#951;SSTD vs. saturation time at different temperatures. The figure shows the plot for N,N-dimethylacetamide and the table with the obtained rates constants and relaxation times. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig17large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 18" src="/files/ftp_upload/54499/54499fig18.jpg" /> 
Figure 18: Eyring plot. The figure shows the plot for N,N-dimethylacetamide. <a href="http://cloudflare.jove.com/files/ftp_upload/54499/54499fig18large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">tsat (sec)</td>
			<td>&#951;SSTD&#160;</td>
			<td>&#951;SSTD&#160;</td>
			<td>&#951;SSTD&#160;</td>
			<td>&#951;SSTD&#160;</td>
			<td>&#951;SSTD&#160;</td>
			<td>&#951;SSTD&#160;</td>
			<td>&#951;SSTD&#160;</td>
		</tr>
		<tr>
			<td>(T = 278 K)</td>
			<td>(T = 283 K)</td>
			<td>(T = 285.5 K)</td>
			<td>(T = 288 K)</td>
			<td>(T = 290.5 K)</td>
			<td>(T = 293 K)</td>
			<td>(T = 295.5 K)</td>
		</tr>
		<tr>
			<td>40</td>
			<td>0.2526</td>
			<td>0.3957</td>
			<td>0.4671</td>
			<td>0.5461</td>
			<td>0.626</td>
			<td>0.6969</td>
			<td>0.7535</td>
		</tr>
		<tr>
			<td>20</td>
			<td>0.2526</td>
			<td>0.3957</td>
			<td>0.4671</td>
			<td>0.5461</td>
			<td>0.626</td>
			<td>0.6969</td>
			<td>0.7535</td>
		</tr>
		<tr>
			<td>10</td>
			<td>0.2383</td>
			<td>0.3806</td>
			<td>0.4537</td>
			<td>0.5355</td>
			<td>0.6199</td>
			<td>0.6969</td>
			<td>0.7535</td>
		</tr>
		<tr>
			<td>5</td>
			<td>0.1904</td>
			<td>0.3193</td>
			<td>0.3919</td>
			<td>0.481</td>
			<td>0.5734</td>
			<td>0.6638</td>
			<td>0.7318</td>
		</tr>
		<tr>
			<td>2.5</td>
			<td>0.1263</td>
			<td>0.2204</td>
			<td>0.2812</td>
			<td>0.3589</td>
			<td>0.4449</td>
			<td>0.5461</td>
			<td>0.626</td>
		</tr>
		<tr>
			<td>1.25</td>
			<td>0.0761</td>
			<td>0.1353</td>
			<td>0.171</td>
			<td>0.2247</td>
			<td>0.2868</td>
			<td>0.3732</td>
			<td>0.4449</td>
		</tr>
		<tr>
			<td>0.625</td>
			<td>0.0467</td>
			<td>0.0739</td>
			<td>0.099</td>
			<td>0.1327</td>
			<td>0.171</td>
			<td>0.2291</td>
			<td>0.2758</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>0.0238</td>
			<td>0.044</td>
			<td>0.0472</td>
			<td>0.0644</td>
			<td>0.0847</td>
			<td>0.1169</td>
			<td>0.1463</td>
		</tr>
	</tbody>
</table>
Table 1: Values of &#951;SSTD. The table shows the values obtained at different saturation times for N,N-dimethylacetamide in the range of temperatures 278-295.5 K.
<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col span="5" />
	</colgroup>
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:145px;">Method</td>
			<td style="width:88px;">SSTD NMR&#160;</td>
			<td rowspan="2" style="width:88px;">SST NMR31</td>
			<td style="width:88px;">Line-shape analysis</td>
			<td style="width:88px;">Line-shape analysis</td>
			<td style="width:88px;">Line-shape analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:145px;">Parameter</td>
			<td style="width:88px;">(This work)</td>
			<td style="width:88px;">(1H NMR)4</td>
			<td style="width:88px;">(1H NMR)5</td>
			<td style="width:88px;">(13C NMR)6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Ea 298 (KJ mol-1)</td>
			<td style="width:88px;">79.7 &#177; 0.1</td>
			<td style="width:88px;">73.1 &#177; 1.4</td>
			<td style="width:88px;">70.5 &#177; 1.7</td>
			<td style="width:88px;">82.0 &#177; 1.3</td>
			<td style="width:88px;">79.5 &#177; 0.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">&#916;H&#8225; (KJ mol-1)</td>
			<td style="width:88px;">77.2 &#177; 0.1</td>
			<td style="width:88px;">70.6 &#177; 1.4</td>
			<td style="width:88px;">68</td>
			<td style="width:88px;">79.5 &#177; 0.4</td>
			<td style="width:88px;">76.6 &#177; 0.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">&#916;S&#8225; (J mol-1 K-1)</td>
			<td style="width:88px;">11.5 &#177; 0.4</td>
			<td style="width:88px;">-10.5 &#177; 5.0</td>
			<td style="width:88px;">-15.0 &#177; 5.1</td>
			<td style="width:88px;">13 &#177; 8</td>
			<td style="width:88px;">3 &#177; 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">&#916;G&#8225;298 (KJ mol-1)</td>
			<td style="width:88px;">73.8 &#177; 0.1</td>
			<td style="width:88px;">73.7 &#177; 2.0</td>
			<td style="width:88px;">72.5</td>
			<td style="width:88px;">75.3 &#177; 0.4</td>
			<td style="width:88px;">75.7 &#177; 0.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:145px;">Solvent</td>
			<td style="width:88px;">Tol-d8</td>
			<td style="width:88px;">Tol-d8</td>
			<td style="width:88px;">CCl4</td>
			<td style="width:88px;">Acetone-d6</td>
			<td style="width:88px;">neat</td>
		</tr>
	</tbody>
</table>
Table 2: Activation parameters. The table shows the activation parameters for the internal rotation of N,N-dimethylacetamide obtained by the SSTD NMR method compared with the same parameters obtained using different NMR methods for analysis.4,5,6 Errors in this table refer to standard deviation errors (SD). (Reproduced from reference21 with permission from the Royal Society of Chemistry).

Watch this Scientific Journal Video about Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes at JoVE.com

Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our ...

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17.7K 조회수.  University of East Anglia. A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.

비디오: Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

연구

생화학

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

화학

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

사이트 감독 스핀 라벨 및 펜타 머 리간드 - 게이 티드 이온 채널의 EPR 분광 연구

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

전자 상자성 공명 영상을 이용하여 다공성 미디어에서 고객 분자의 확산의 제자리 모니터링에

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

15 Μs-ms 타임스케일에 단백질 형성 역학의 조사를 위한 N CPMG 이완 분산

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA의 1 H R 1θ 이완 분산 실험의 샘플 준비 및 설치의 실제적인 양상

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

빠른 스캔 전자 스핀 공명 이미징 생리 학적으로 중요 매개 변수에 대한 새로운 길을 엽니 다<em&gt; 생체</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

생체공학

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

글라이칸-단백질 상호 작용 분석을 위한 고급 NMR 기술

Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

저자 스포트라이트: Unveiling the Structural and Dynamic Aspects of Glycan Molecular Recognition (글라이칸 분자 인식의 구조적 및 동적 측면 공개)

Here, we present a protocol detailing the acquisition, processing, and analysis of a series of NMR experiments aimed at characterizing protein-glycan interactions in solution. Most common ligand-based and protein-based methodologies are outlined, which undoubtedly contribute to the fields of structural glycobiology and molecular recognition studies.

글라이칸-단백질 상호 작용 해체: 핵 자기 공명(NMR)을 위한 구조

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and ...

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

사이트 감독 스핀 라벨 및 펜타 머 리간드 - 게이 티드 이온 채널의 EPR 분광 연구

전자 상자성 공명 영상을 이용하여 다공성 미디어에서 고객 분자의 확산의 제자리 모니터링에

¹⁵ Μs-ms 타임스케일에 단백질 형성 역학의 조사를 위한 N CPMG 이완 분산

RNA의 1 H R _1θ 이완 분산 실험의 샘플 준비 및 설치의 실제적인 양상

빠른 스캔 전자 스핀 공명 이미징 생리 학적으로 중요 매개 변수에 대한 새로운 길을 엽니 다

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

글라이칸-단백질 상호 작용 해체: 핵 자기 공명(NMR)을 위한 구조

글라이칸-단백질 상호 작용 해체: 핵 자기 공명(NMR)을 위한 구조