We thank Drs. Jinfa Ying and Kristin Cano for helpful discussions and technical assistance. DSL is a St. Baldrick&#39;s Scholar and acknowledges the support of the St. Baldrick&#39;s Foundation (634706). This work was supported in part by the Welch Foundation (AQ-2001-20190330) to DSL, the Max and Minnie Tomerlin Voelcker Fund (Voelcker Foundation Young Investigator Grant to DSL), UTHSA Start-Up Funds to DSL, and a Greehey Graduate Fellowship in Children&#39;s Health to CNJ. This work is based upon research conducted in the Structural Biology Core Facilities, part of the Institutional Research Cores at the University of Texas Health Science Center at San Antonio supported by the Office of the Vice President for Research and the Mays Cancer Center Drug Discovery and Structural Biology Shared Resource (NIH P30 CA054174).

General requirements for the protocol: protein purification facilities, UV-Vis spectrometer, high-field NMR spectrometer and operating software, post-processing analysis software including; NMRPipe32, Sparky33, (or CCPN Analysis34, or NMRViewJ35).
1. Recombinant expression and purification of a protein for PRE measurements
<ol>
	<li>Design an expression construct for the protein of interest so tha...

All authors have read and approved the manuscript. No conflicts of interest are declared.

A method for characterizing transient interactions that exist at low populations between intrinsically disordered proteins and various binding partners using PRE has been presented. In the example shown, the protein is self-associating, and thus the PRE may arise from a combination of inter and intramolecular interactions. This method is readily extended to heterogeneous samples where the interactions between two different proteins may be characterized. Complementary information about how different regions of the protein...

Intrinsic disorder (ID) describes proteins (IDPs) or regions within proteins (IDRs) that do not spontaneously fold into stable secondary or tertiary structures but are biologically active. Generally, the function of IDP/IDRs is to facilitate specific yet reversible interactions with biomolecules at physiological conditions1. Thus, IDPs and IDRs are involved in a range of cellular functions, including recruitment, organization, and stabilization of multi-protein complexes, for example, the assembly and activity of the spliceosome2, recruitment and organization of components at sites of DNA damage3, organization, and stabilization of the recruitment of transcription complexes4, or of the chromatin remodeler BAF5. Additionally, IDPs are found at signaling nexuses where their promiscuity for different binding partners enables them to mediate information transfer through cellular protein networks6. Recent work has also revealed a proclivity for IDR regions to self-associate forming biomolecular condensates through the process of liquid-liquid phase separation7. Many of the aforementioned functions involving ID are also now thought to involve some aspect of condensate formation8. Despite the importance of ID for biomolecular complex assembly, stabilization, scaffolding, and signal transduction, the atomic details of their specific interactions are difficult to identify since IDPs and IDRs are typically not amenable to structural investigations using x-ray crystallography or cryogenic electron microscopy.
Nuclear magnetic resonance (NMR) is an ideal technique for investigating ID as it is not dependent on the presence of rigid or homogenous structural ensembles but reports on the immediate local environment of individual nuclei. The resonant frequency, or chemical shift, of a nucleus in a given molecule is influenced by weak magnetic fields induced by the local electronic distribution, which in turn is dependent on bond lengths, angles, nearness of other nuclei, interactions with binding partners, and other factors9. Thus, each nucleus acts as a unique, site-specific structural probe sensitive to changes in its local chemical environment. Despite these advantages, NMR is a bulk technique, and the observed chemical shift is the average of all the environments sampled by a particular nucleus. A range of NMR techniques, many of which are described in this issue, have been developed to recover structural, dynamic, and kinetic information about high energy, lowly-populated biomolecular conformations contained in the averaged chemical shift10,11. Although transiently populated, identification and quantification of these states are important for determining the details of functional mechanisms12. For example, in the case of IDPs and IDRs, the conformational ensemble may be biased to preferentially sample conformations that are productive for the formation of encounter complexes with physiological binding partners. Detection of these states, as well as identification of the residue-specific inter- and intra-molecular interactions and dynamics, are important to determine the underlying structural mechanisms of protein function and complex formation.
A protocol for using paramagnetic relaxation enhancement (PRE) NMR to investigate transient, lowly-populated states important for the formation of IDP/IDR-mediated biomolecular complexes is described13. This approach is useful for studying the transient protein-protein interactions such as those that promote the assembly of amyloid fibrils from &#945;-synuclein14,15 or the self-association of FUS16, as well as to characterize specific protein-protein interactions such as between signaling proteins17. An example of a self-associating IDP is presented, where specific inter- and intra-molecular interactions result in preferentially compacted states as well as site-specific interactions that drive self-association.
The PRE arises from the magnetic dipolar interaction of a nucleus to a paramagnetic center with an isotropic g-tensor, commonly supplied in the form of an unpaired electron on a nitroxide group or as a paramagnetic metal atom18 (Figure 1). While atoms with anisotropic g-tensors also produce a PRE effect, analysis of these systems is more difficult due to confounding effects contributed by the pseudo-contact shifts (PCS) or residual dipolar coupling (RDC)13,19. The strength of the interaction between a nucleus and the paramagnetic center is dependent on the &#60;r-6&#62; distance between the two. This interaction results in an increase in nuclear relaxation rates, which causes detectable line broadening even for long-range interactions (~10-35 &#197;), because the magnetic moment of the unpaired electron is so strong20,21. Detection of transient states with the PRE is possible if the following two conditions are met; (1) the transient interaction is in fast exchange on the NMR timescale (observed chemical shift is a population-weighted average of the exchanging states); and (2) the nuclei to paramagnetic center distance is shorter in the transiently populated state than in the major state11. The transverse PRE is denoted &#915;2 and, for practical purposes, is calculated from the difference in 1H transverse relaxation rates between a sample containing a paramagnetic center and a diamagnetic control. For an in-depth treatment of the theory of the PRE and related pseudocontact shifts in fast and slow exchange regimes, the reader is referred to the comprehensive reviews by Clore and co-workers13,22. Here, only the situation where 1HN-&#915;2 is in the fast exchange regime is considered, where because of the r-6-dependence of the PRE, the observed relaxation rate is related to both the distance to which the paramagnetic center approaches the nucleus as well as the amount of time it spends in that conformation. Therefore, transient conformations that do not involve a close approach produce a small PRE while closer interactions, even if short-lived, will produce a larger PRE.
For IDPs, the PRE is used to measure and differentiate the interactions occurring within a single molecule (intramolecular) and between separate molecules (intermolecular). By attaching a paramagnetic center to an NMR visible (e.g., 15N-labeled) or NMR invisible (e.g., natural abundance 14N) protein, the source (inter- or intra-molecular) of the PRE may be determined (Figure 2). Site-directed mutagenesis that introduces a cysteine residue is a convenient approach to attach a paramagnetic center (spin-label) to a protein23. Several types of molecules have been proposed for use as spin labels, including metal chelating (EDTA-based) and free-radical (nitroxide-based)24. Various nitroxide spin labels have been described and are available with different cysteine-reactive chemistries such as methanethiosulphonate, maleimide, and iodoacetamide25,26 (Figure 1). Inherent flexibility of the tag or of the linker may be problematic for certain analyses, and in these situations, different strategies have been proposed to limit the motion of the tag, such as by adding bulky chemical groups or the use of a second linker to anchor the tag to the protein (two site attachment)27,28. Additionally, commercially available tags may contain diastereomeric proteins but generally this will not contribute to the observed PRE29. The use of the 3-Maleimido-PROXYL attached to a free cysteine via maleimide chemistry is described since it is readily available, cost-effective, non-reversible, and the reducing agent tris(2-carboxyethyl)phosphine (TCEP) can be maintained in the solution throughout the labeling reaction. Since 3-Maleimido-PROXYL has an isotropic g-tensor, no PCS or RDCs are induced, and the same chemical shift assignments can be used for both the paramagnetic and diamagnetic samples13.
The 1HN-T2 is measured using a two time-point strategy (Ta, Tb) that has previously been shown to be as accurate as collecting a full evolution series consisting of 8 to 12 time points30. The first time point (Ta) is set as close to zero as practical, and the optimal length of the second time point is dependent on the magnitude of the largest expected PRE for a given sample and can be estimated from: Tb ~ 1.15/(R2,dia + &#915;2) where R2,dia represents the R2 of the diamagnetic sample13. If the magnitude of the largest PREs is unknown, setting Tb to ~ one times the 1H T2 of the protein is a good initial estimate and further optimized by adjusting T2 to improve the signal to noise. This two-point measurement strategy significantly reduces the experimental time required to measure PREs and allows time for more signal averaging, particularly since relatively dilute samples are used to minimize the effects of non-specific contacts between molecules. An HSQC-based pulse sequence is used to measure 1HN-T2 and has been described in detail elsewhere30. For improved sensitivity, the hard pulses of the forward and back INEPT transfers may be replaced with shaped pulses; alternatively, the sequence is readily converted to a TROSY-based readout31. Since IDPs typically have much longer transverse relaxation rates resulting in narrower line widths (due to the inherent disorder) than similarly sized globular proteins, long acquisition times in the indirect dimension may be used to improve spectral resolution and alleviate the chemical shift dispersion limitation inherent for IDPs.
PRE is a useful tool for studying protein-protein and protein-nucleic acid interactions, particularly interactions that are transient or lowly populated. A detailed protocol for the preparation of an NMR sample suitable for measuring PREs, including steps for protein purification, site-directed spin labeling, setting up and calibrating the pulse program, processing, and interpreting the NMR data, is provided. Important experimental considerations are noted throughout that may impact data quality and experimental outcome, including sample concentration, selection of the spin-label, and removal of paramagnetic components.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#181;m and 0.22 &#181;m syringe filters</td>
			<td>Millipore Sigma</td>
			<td>SLHVM33RS 
			SLGVR33RS</td>
			<td>Filter lysate before first purification step and before size exclusion chromatography.</td>
		</tr>
		<tr>
			<td>100 mm Petri Dish</td>
			<td>Fisher</td>
			<td>FB0875713</td>
			<td>Agar plates for bacterial transformation.</td>
		</tr>
		<tr>
			<td>14N Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>576794</td>
			<td>Use of 15N in M9 medium will produce an NMR visible protein, 14N will produces an NMR invisible protein</td>
		</tr>
		<tr>
			<td>15N Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>299251</td>
			<td>Use of 15N in M9 medium will produce an NMR visible protein, 14N will produces an NMR invisible protein</td>
		</tr>
		<tr>
			<td>3 L Fernbach baffled flask</td>
			<td>Corning</td>
			<td>431523</td>
			<td>Bacterial expression culture</td>
		</tr>
		<tr>
			<td>3-Maleimido-Proxyl</td>
			<td>Sigma Aldrich</td>
			<td>253375</td>
			<td>Nitroxide spin label</td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Thermo Fisher</td>
			<td>14-432-22</td>
			<td>Solution/protein storage</td>
		</tr>
		<tr>
			<td>Amicon centrifugal filter</td>
			<td>Millipore Sigma</td>
			<td>UFC900308</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Ampicillan</td>
			<td>Sigma Aldrich</td>
			<td>A5354</td>
			<td>Antibiotic for a selective marker, exact choice depends on the expression construct plasmid</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Oahus</td>
			<td>30061978</td>
			<td>Explorer Pro, for weighing reagents</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>AX1775</td>
			<td>Reduces nitroxide spin label</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td>Sterilize glassware and culture media</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>C4901</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>Centrifuge bottles</td>
			<td>Thermo Fisher</td>
			<td>010-1459</td>
			<td>Harvest E. coli cells after recombinant protein expression</td>
		</tr>
		<tr>
			<td>Centrifuge, hand-crank</td>
			<td>Thomas Scientific</td>
			<td>0241C68</td>
			<td>Boekel hand-driven, low-speed centrifuge with 15 mL buckets that can accommodate NMR tubes</td>
		</tr>
		<tr>
			<td>Chelex 100</td>
			<td>Sigma Aldrich</td>
			<td>C7901</td>
			<td>Remove contaminating paramagnetic compounds from buffer solutions</td>
		</tr>
		<tr>
			<td>Computer workstation</td>
			<td></td>
			<td></td>
			<td>Linux or Mac OS compatable with NMR data processing and analysis software packages such as NMRPipe and Sparky</td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Sigma Aldrich</td>
			<td>151882</td>
			<td>Needed for NMR lock signal</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>D9434</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>Dibasic Sodium Phosphate</td>
			<td>Sigma Aldrich</td>
			<td>S5136</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>Ellman&#39;s reagent (5,5-dithio-bis-(2-nitrobenzoic acid)</td>
			<td>Thermo Fisher</td>
			<td>22582</td>
			<td>Quantification of free cystiene residues</td>
		</tr>
		<tr>
			<td>High speed centrifuge tubes</td>
			<td>Thermo Fisher</td>
			<td>3114-0050</td>
			<td>Used to clear bacterial lysate.</td>
		</tr>
		<tr>
			<td>High-field NMR instrument (600 - 800 MHz)</td>
			<td>Bruker</td>
			<td></td>
			<td>Equiped with a multichannel cryogenic probe and temperature control</td>
		</tr>
		<tr>
			<td>IMAC column, HisTrap FF</td>
			<td>Cytvia</td>
			<td>17528601</td>
			<td>Initial fractionation of crude bacterial lysate</td>
		</tr>
		<tr>
			<td>Isopropyl B-D-thiogalactoside (IPTG)</td>
			<td>Sigma Aldrich</td>
			<td>I6758</td>
			<td>Induces protein expression for genes under control of lac operator</td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Thermo Fisher</td>
			<td>22700025</td>
			<td>Items are used for transforming E. coli to express protein of interest, substitions for any of these items with like products is acceptable.</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Thermo Fisher</td>
			<td>12780052</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-pressure chromatography system</td>
			<td>Bio-Rad</td>
			<td>7318300</td>
			<td>BioRad BioLogic is used for low-pressure chomatograph such as running IMAC columns</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>Medium pressure chromatography system</td>
			<td>Bio-Rad</td>
			<td>7880007</td>
			<td>BioRad NGC equipped with a multi-wavelength detector, pH and conductivity monitors, and automatic fraction collector</td>
		</tr>
		<tr>
			<td>MEM vitamin solution</td>
			<td>Sigma Aldrich</td>
			<td>M6895</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>Microfluidizer</td>
			<td>Avestin</td>
			<td>EmulsiFlex-C3</td>
			<td>Provides rapid and efficient bacterial cell lysis</td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>Calibrated set of micropippetters with properly fitting disposable tips (available from multiple manufacturers e.g. Eppendorf)</td>
		</tr>
		<tr>
			<td>Monobasic potassium phosphate</td>
			<td>Sigma Aldrich</td>
			<td>1551139</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>NMR pipettes</td>
			<td>Sigma Aldrich</td>
			<td>255688</td>
			<td>To remove sample from NMR tube</td>
		</tr>
		<tr>
			<td>NMR sample tube</td>
			<td>NewEra</td>
			<td>NE-SL5</td>
			<td>Suitable for high-field NMR spectrometers</td>
		</tr>
		<tr>
			<td>Preparative Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti J-HC</td>
			<td>Harvest E. coli cells after recombinant protein expression</td>
		</tr>
		<tr>
			<td>Round bottom polystyrene centrifuge tubes</td>
			<td>Corning</td>
			<td>352057</td>
			<td>Clear bacterial lysate</td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Eppendorf</td>
			<td>S44I200005</td>
			<td>Temperature controlled growth of E. coli starter and expression cultures</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S5886</td>
			<td>M9 media component</td>
		</tr>
		<tr>
			<td>Sonicating water bath and vacuum source</td>
			<td>Thomas Scientific</td>
			<td></td>
			<td>Used to degas buffer solutions</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Thermo Fisher</td>
			<td>FB505110</td>
			<td>Used for bacterial cell lysis or shearing bacterial DNA</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Implen</td>
			<td>OD600 Diluphotometer</td>
			<td>Monitor growth of E.coli protein expression cultures</td>
		</tr>
		<tr>
			<td>Superdex 200 16/600 size exculsion colum</td>
			<td>Cytvia</td>
			<td>28989333</td>
			<td>Final protein purification step</td>
		</tr>
		<tr>
			<td>Topspin software, version 3.2 or later</td>
			<td>Bruker</td>
			<td></td>
			<td>Operating software for the NMR instrument</td>
		</tr>
		<tr>
			<td>Transformation competent E. coli cells</td>
			<td>Thermo Fisher</td>
			<td>C600003</td>
			<td>One Shot BL21 Star (DE3) chemically competent E. coli, other strains may be compatable</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td>ThermoFisher</td>
			<td>20490</td>
			<td>Reducing agent compatable with some sulfhydryl-reactive conjugations</td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>Implen</td>
			<td>NP80</td>
			<td>Measure protein concentration.</td>
		</tr>
		<tr>
			<td>Water bath, temperature controlled</td>
			<td>ThermoFisher</td>
			<td>FSGPD25</td>
			<td>For heat shock step of bacterial transformation</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma Aldrich</td>
			<td>Y1625</td>
			<td>For supplementing M9 media if required</td>
		</tr>
	</tbody>
</table>

paramagnetic relaxation enhancement for detecting and characterizing self-associations of intrinsically disordered proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

Intramolecular 1HN-&#915;2 PREs were recorded on a self-associating, intrinsically disordered fragment (residues 171-264) derived from the low-complexity domain of the RNA-binding protein EWSR142 (Figure 3). Residues in close sequential proximity to the spin-label attachment point (e.g., residue 178 or 260 in Figure 3) are expected to be significantly broadened and are not detectable in the spectr...

Watch this Scientific Journal Video about Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins at JoVE.com

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

The paramagnetic relaxation enhancement technique can be utilized to characterize and measure intermolecular distances and quantify transient and/or lowly populated biomolecular interactions. Here, we have applied this technique to capture interactions that precede protein condensation. The sensitivity of PREs enables atomic resolution identification, detection, and quantification of dynamic state that remain invisible and inaccessible to other NMR techniques.Key steps in this protocol include removing the unconjugated nitroxide spin label and carefully analyzing the spectra to accurately measure peak height. Additionally, caution must be exercised during NMR experiment setup and pulse calibration. To begin, add 3-maleimido-PROXYL from a stock solution at 20 times molar excess of the 15N isotopically labeled protein of interest.Incubate overnight at room temperature or four degrees Celsius protected from light and oxygen and with gentle rocking or nutation. The following day, remove the non-reacted free spin label to prevent non-specific solvent PRE using either extensive dialysis of the protein sample or using gel filtration. Then use Ellman's reagent to quantify free sulfhydryl groups in the solution.Measure the absorbance using a microplate reader and construct the standard curve. To measure intramolecular PRE, prepare 15N isotopically enriched spin labeled protein to a concentration of at least 100 micromolar, but not more than 300 micromolar, in a buffer suitable for NMR. Then using a long stem glass pipette or micropipette, transfer the NMR sample to a five millimeter NMR tube appropriate for use in high-field magnets.Place the sample in the magnet. Lock on the deuterium signal using the lock command and attune and match the proton channel according to facility protocols. Then adjust the shims using the top shim subroutine to optimize solvent signal suppression.Next, calibrate the proton pulse using the P-OPT program. Then calibrate the 15N pulse against a standard sample. Determine the correct attenuation for shaped pulses using the shape tool subroutine.Then open the appropriate pulse shape file by clicking the folder icon. The shaped pulses are found in the pulse parameters section of acquisition parameters. After loading the pulse definition file, click Analyze Waveform, followed by Integrate Shape.Input the calibrated proton 90 degree hard pulse, desired shape pulse length, and rotation. Then calculate the power level of the shaped pulse by adding the change of power level to the attenuation for the calibrated 90 degree pulse. Record a standard proton 15N HSQC to optimize sweep width, carrier frequency, and check water suppression.Finally, adjust the sweep width and the number of indirect dimension increments using the SW and TD commands or directly in the appropriate dialog boxes. Calibrate the shaped pulses as demonstrated previously. Then enter the calibrated pulse lengths in the pulse parameters section on the Acquisition Parameters tab.To measure the amide proton T2 using the two time delay point approach, set the time delays by editing the VD list file. The first delay is set to 0.01 milliseconds. Using the relationship to the expected maximum PRE, choose the second delay.Then determine a suitable value by comparing the first increments of the first and second delay spectra and adjusting the second delay such that the signal decays to between 40 to 50%of its initial value. Determine the number of complex points to record and the number of scans for sufficient signal averaging. Then use the command EXPT to calculate the experiment time and start the experiment with the command ZG.After dissolving sodium ascorbate in the NMR buffer, adjust the pH to match that of the original NMR buffer.Then add the ascorbic acid to the NMR tube at a 10 times molar excess over the concentration of the spin label by placing a droplet below the rim of the tube. Cap the tube and carefully invert to mix. Spin at 200 to 400 G for 10 to 20 seconds in a hand cranked centrifuge to settle the sample at the bottom of the tube.After wrapping the tube in foil, allow the reaction to proceed for at least three hours. Then record amide proton T2 on the diamagnetic sample using the same parameters used for the paramagnetic sample. Intramolecular amide proton gammas PREs were recorded on a self-associating intrinsically disordered fragment derived from the low complexity domain of the RNA binding protein EWSR1.Residues in close sequential proximity to the spin label attachment point are expected to be significantly broadened and are not detectable in the spectrum. Residues that were sequentially spaced from the attachment point yet showed enhanced gamma were spatially close to the spin label. In the case of intrinsically disordered proteins, recording and analyzing purees will yield information about intramolecular contacts and transient interaction surfaces.Combining PRE measurements with other biophysical methods such as dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and computational methods can provide deeper insights. PRE measurement aids in characterizing transient sparsely populated state. This approach could be expanded to characterize interactions important for myriad biological processes such as molecular recognition, allosteric conformational selection, and assembly processes including assembling membraneless organelles via phase separation.

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Research

JoVE Journal

Biochemistry

1.6K Görüntüleme.  The University of Texas Health Science Center at San Antonio. Paramanyetik gevşeme geliştirme tekniği, moleküller arası mesafeleri karakterize etmek ve ölçmek ve geçici ve/veya düşük nüfuslu biyomoleküler etkileşimleri ölçmek için kullanılabilir. Burada, protein yoğunlaşmasından önceki etkileşimleri yakalamak için bu tekniği uyguladık. PRE'lerin hassasiyeti, diğer NMR teknikleri tarafından görünmez ve erişilemez kalan dinamik durumun atomik çözünürlüğün tanımlanmasını, algılanmasını ve ölçülmesini sağlar.