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).

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><tbody><tr><td>0.45 &amp;micro;m and 0.22 &amp;micro;m syringe filters</td><td>Millipore Sigma</td><td>SLHVM33RS&lt;br/&gt; 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>&lt;sup&gt;14&lt;/sup&gt;N Ammonium chloride</td><td>Sigma Aldrich</td><td>576794</td><td>Use of &lt;sup&gt;15&lt;/sup&gt;N in M9 medium will produce an NMR visible protein,&lt;sup&gt; 14&lt;/sup&gt;N will produces an NMR invisible protein</td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;N Ammonium chloride</td><td>Sigma Aldrich</td><td>299251</td><td>Use of &lt;sup&gt;15&lt;/sup&gt;N in M9 medium will produce an NMR visible protein,&lt;sup&gt; 14&lt;/sup&gt;N 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&#039;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 ...

paramagnetic-relaxation-enhancement-for-detecting-characterizing-self

Research

JoVE Journal

Biochemistry

1.7K Views.  The University of Texas Health Science Center at San Antonio. De paramagnetische relaxatieverbeteringstechniek kan worden gebruikt om intermoleculaire afstanden te karakteriseren en te meten en voorbijgaande en / of laagbevolkte biomoleculaire interacties te kwantificeren. Hier hebben we deze techniek toegepast om interacties vast te leggen die voorafgaan aan eiwitcondensatie. De gevoeligheid van PREs maakt identificatie van atomaire resolutie, detectie en kwantificering van dynamische toestanden mogelijk die onzichtbaar en ontoegankelijk blijven voor a...

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

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engineering

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

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 ...

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.

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

Chemistry

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

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.

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

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

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.

Author Spotlight: Unveiling the Structural and Dynamic Aspects of Glycan Molecular Recognition

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.

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

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Bioengineering

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

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

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