Site-specific Spin Labeling Research Articles

Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies.

Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems.Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.

Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1.

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.

Disassembly/reassembly strategy for the production of highly pure GroEL, a tetradecameric supramolecular machine, suitable for quantitative NMR, EPR and mutational studies

GroEL, a prototypical member of the chaperonin class of chaperones, is a large supramocular machine that assists protein folding and plays an important role in proteostasis. GroEL comprises two heptameric rings, each of which encloses a large cavity that provides a folding chamber for protein substrates. Many questions remain regarding the mechanistic details of GroEL facilitated protein folding. Thus, data at atomic resolution of the type provided by NMR and EPR are invaluable. Such studies often require complete deuteration of GroEL, uniform or residue specific 13C and 15N isotope labeling, and the introduction of selective cysteine mutations for site-specific spin labeling. In addition, high purity GroEL is essential for detailed studies of substrate-GroEL interactions as quantitative interpretation is impossible if the cavities are already occupied and blocked by other protein substrates present in the bacterial expression system. Here we present a new purification protocol designed to provide highly pure GroEL devoid of non-specific protein substrate contamination.

Dynamically polarized samples for neutron protein crystallography at the Spallation Neutron Source

To prepare for the next generation neutron scattering instruments for the planned second target station at the Spallation Neutron Source (SNS) and to broaden the scientific impact of neutron protein crystallography at the Oak Ridge National Laboratory, we have recently ramped up our efforts to develop a dynamically polarized target for neutron protein crystallography at the SNS. Proteins contain a large amount of hydrogen which contributes to incoherent diffraction background and limits the sensitivity of neutron protein crystallography. This incoherent background can be suppressed by using polarized neutron diffraction, which in the same time also improves the coherent diffraction signal. Our plan is to develop a custom Dynamic Nuclear Polarization (DNP) setup tailored to neutron protein diffraction instruments. Protein crystals will be polarized at a magnetic field of 5 T and temperatures of below 1 K. After the dynamic polarization process, the sample will be brought to a frozen-spin mode in a 0.5 T holding field and at temperatures below 100 mK. In a parallel effort, we are also investigating various ways of incorporating polarization agents needed for DNP, such as site specific spin labels, into protein crystals.

Bacterial Chemoreceptor Dynamics: Helical Stability in the Cytoplasmic Domain Varies with Functional Segment and Adaptational Modification

Dynamics are thought to be important features of structure and signaling in the cytoplasmic domain of bacterial chemoreceptors. However, little is known about which structural features are dynamic. For this largely helical domain, comprising a four-helix bundle and an extended four-helix coiled coil, functionally important structural dynamics likely involves helical mobility and stability. To investigate, we used continuous wave EPR spectroscopy and site-specific spin labels that directly probed, in essentially physiological conditions, the mobility of helical backbones in the cytoplasmic domain of intact chemoreceptor Tar homodimers inserted into lipid bilayers of Nanodiscs. We observed differences among functional regions, between companion helices in helical hairpins of the coiled coil and between receptor conformational states generated by adaptational modification. Increased adaptational modification decreased helical dynamics while preserving dynamics differences among functional regions and between companion helices. In contrast, receptor ligand occupancy did not have a discernable effect on dynamics to which our approach was sensitive, implying that the two sensory inputs alter different chemoreceptor features. Spectral fitting indicated that differences in helical dynamics we observed for ensemble spin-label mobility reflected differences in proportions of a minority receptor population in which the otherwise helical backbone was essentially disordered. We suggest that our measurements provided site-specific snapshots of equilibria between a majority state of well-ordered helix and a minority state of locally disordered polypeptide backbone. Thus, the proportion of polypeptide chain that is locally and presumably transiently disordered is a structural feature of cytoplasmic domain dynamics that varies with functional region and modification-induced signaling state.

Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568–596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574–589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.

Application of Site-Specific Spin Labeling for NMR Detecting Inhibitor-Induced Conformational Change of HIV-1 Reverse Transcriptase.

Paramagnetism-assisted nuclear magnetic resonance (NMR) techniques can provide long-range structural information complemented with local information derived from chemical-shift perturbation and nuclear Overhauser effect data. Here, we address the application of paramagnetic relaxation enhancement (PRE) to detect inhibitor-induced conformational change of a drug target protein using human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) as a model protein. Using a site-specific spin-labeled HIV-1 RT mutant with selective (13) C labeling, conformation-dependent PREs were successfully observed reflecting the stabilization of an open conformation of this enzyme caused by inhibitor binding. This study demonstrates that the paramagnetism-assisted NMR approach offers an alternative strategy in protein-based drug screening to identify allosteric inhibitors of a target protein.

Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau

Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255-441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50-70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Mode of substrate recognition by the Josephin domain of ataxin-3, which has an endo-type deubiquitinase activity

Ataxin-3, which is encoded by a gene that has been associated with Machado–Joseph disease, contains a catalytic N-terminal Josephin domain with deubiquitinase activity. Here, we show that the Josephin domain of ataxin 3 catalyzes endo-type cleavage of Lys48-linked polyubiquitin. Furthermore, NMR data obtained following site-specific paramagnetic spin labeling of Lys48-linked di-ubiquitin revealed that both ubiquitin units interact with the Josephin domain, with the C-terminal Gly76 of the proximal unit being situated in the vicinity of the catalytic triad of Josephin domain. Our results help to elucidate how the substrate is recognized by the Josephin domain and properly positioned for an endo-type deubiquitination reaction.

Residue specific partitioning of KL4 into phospholipid bilayers

KL4, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL4 and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL4 on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL4, and the partitioning depths of specific KL4 residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL4 induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL4 (V.C. Antharam et al., 2009), we are able to distinguish how KL4 affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL4 also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL4 are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.

Inhibition of energy transduction in P‐glycoprotein (974.3)

Cancer chemotherapy failures often involve the over‐expression of ABC‐transporters like the MDR1 P‐glycoprotein (P‐gp). These transporters normally protect the cell by pumping toxins and xenobiotics across the plasma membrane. Selection for cells over‐expressing P‐gp during chemotherapy, however, results in populations that are drug resistant. Recently, we used massively parallel in silico drug docking to identify molecules predicted to interact with higher affinity at the energy transducing structures of P‐gp than at the drug transporting structures. In vitro assays verified that the compounds inhibited verapamil‐stimulated ATP hydrolysis and only marginally stimulated basal ATP hydrolysis activities. This indicates that these compounds do not interact with drug binding domains. The effects of the identified compounds on ATP binding was then studied using spin‐labeled ATP analogs and ESR spectroscopy. Results showed that some of the identified compounds decreased ATP binding stoichiometry to P‐gp while others decreased ATP binding affinities. These studies further showed that inhibition by the compounds was reversible. The investigations suggest that the discovered P‐gp inhibitors may make good drug leads. Experiments using ESR spectroscopy and site‐specific spin‐labeling of the drug binding domain are currently underway that may shed further light on the molecular mechanism of inhibition.Grant Funding Source: NIGMS1R15GM094771 ‐ 01A1, Communities Foundations of Texas

In-Situ Spin Labeling of His-Tagged Proteins: Distance Measurements under In-Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin-labeled chemical recognition unit for switchable and concomitantly high affinity binding to His-tagged proteins was synthesized. In combination with an orthogonal site-directed spin label, this novel spin probe, Proxyl-trisNTA (P-trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate-binding protein, 2) its substrate-dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site-specific spin labeling in cell lysates under in-cell conditions. This approach will open new avenues towards in-cell EPR.

Orthogonal Spin Labeling and Gd(III)–Nitroxide Distance Measurements on Bacteriophage T4-Lysozyme

We present the first example of chemoselective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a gadolinium(III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity.

Paramagnetic Relaxation Enhancement Reveals Oligomerization Interface of a Membrane Protein

Protein-protein interactions play critical roles in cellular function and oligomerization of membrane proteins is a commonly observed phenomenon. Determining the oligomerization state and defining the intermolecular interface in the bilayer is generally a difficult task. Here, we use site-specific spin labeling to demonstrate that relaxation enhancements induced by covalently attached paramagnetic tag can provide distance restraints defining the intermonomer interface in oligomers formed by a seven-helical transmembrane protein Anabaena Sensory Rhodopsin (ASR). We combine these measurements with visible CD spectroscopy and cross-linking experiments to demonstrate that ASR forms tight trimers in both detergents and lipids.

Glass Transition Temperature and β Relaxation Temperature around Chain End of Polystyrene Determined by Site Specific Spin Labeling

A glass transition temperature, T(g), and a β relaxation temperature, T(β), of spin-labeled polystyrene (PS) having a number average molecular weight (M(n)) of ca. 25 kDa were determined by the microwave power saturation (MPS) method of electron spin resonance (ESR). Spin labeling was selectively carried out at chain ends or midchain segments. This method allowed us to determine the local T(g) and the local T(β) around the spin-labeled sites, selectively. The T(g) determined by the ESR, T(g,ESR), was in good agreement with that determined by differential scanning calorimetry, T(g,DSC); the T(g,ESR) decreased with decreasing M(n) with blending oligomers as well as the T(g,DSC). The T(g,ESR) for the end-labeled PS (PS-E) was equal to that for the midchain-labeled PS (PS-M) irrespective of the M(n). However, we previously reported that the PS-E showed distinctly higher segmental mobility than the PS-M in the temperature range 423-463 K (above T(g)). Therefore, we conclude that the chain ends intrinsically have higher segmental mobility than midchain segments due to the discontinuity of repeat units; however, the mobilities of chain ends and midchain segments are averaged out in the vicinity of T(g) due to the cooperativities with neighboring numerous chain segments. Concerning the β relaxation, the T(β) determined by the MPS was in good agreement with those determined by dielectric and dynamic mechanical spectroscopies and dilatometry. The T(β) of the PS-E was the same with that of the PS-M within experimental uncertainties; the T(β) was insensitive to the M(n) in contrast to the T(g). Therefore, we conclude that the effect of chain end is little on the β relaxation of PS due to its local character. In addition, the effect of annealing at 353 K was found to be the same for the T(β)s of the PS-E and PS-M.

Elucidating the Alpha-Synuclein Fibril Fold by Pulsed EPR

Amyloid fibrils are constituents of the plaques that are the hallmarks of neurogdegenerative diseases. In Parkinson's disease, these plaques (Lewy bodies) consist predominantly of the α-synuclein (αS) protein. To understand and interfere with aggregation, the structure of the fibrils (right Fig., green) needs to be known. Here we study the molecular architecture of the fibrils of αS by measuring distances between pairs of residues in the protein using double electron-electron paramagnetic resonance (DEER). Site-specific spin labeling was employed to create nine doubly labeled αS-variants that were investigated in the fibrillar state. Diamagnetic dilution with wild-type αS suppressed intermolecular interactions. The intramolecular distances provide constraints for the fold of the protein inside the fibril. Intramolecular distances were unambiguously observed for four pairs (41/69, 56/69, 56/90 and 69/90). Three of these distances (arrows) provide the constraints to suggest a model for the fold between residues 56 and 90 in the fibril (light blue). Assuming that only parallel β-sheets occur (l.-blue arrows), a model of four adjacent β-strands results (II-V), in which the strands comprise of eight to twelve residues each.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The Subunit B-Dimer of the F1-ATPase Influences Conformational Transitions During Catalysis

The F1FO-ATP synthase, the enzyme responsible for most of ATP generated in biological systems, is a highly asymmetric enzyme complex. Part of the asymmetry stems from interactions of the nucleotide binding subunits α and β with the central rotor subunit gamma. Subunit gamma transfers the energy of the proton-driven rotation of the FO subunit c ring to subunit β, and induces conformational changes that drive ATP release during synthesis. We hypothesize that interactions with the external stalk subunit b-dimer will introduce additional asymmetry to the enzyme complex that may influence catalytic transitions. To investigate our hypothesis, we substituted two tyrosines in subunit β at amino acid positions 331 and 354 for cysteines in an otherwise cysteine-less enzyme. These tyrosines were shown previously to be part of the catalytic and non-catalytic sites, respectively. Using the cysteine-less enzyme as a control, our experiments showed that trapping efficiency of the cysteine-less protein as well as both mutants using AlFx- in what is considered to be a transition-state like conformation was influenced by the presence of soluble b2. Reactivation of trapped enzyme occurred in presence of excess of MgATP and was also influenced by the presence of soluble b2. In separate experiments using site-specific spin labeling and two different spin labels as reporter groups, we observed changes in the conformation of both the catalytic and non-catalytic sites during trapping. In each case, the presence of subunit b-dimer resulted in changes in the conformational transitions. Experiments are underway to localize the binding site of the subunit b-dimer in either the catalytic or the non-catalytic cleft of the ATPase.

Functional Conformational Changes in Lipoxygenase

Structures of catalytic domains of lipoxygenases, including representatives from mammals, corals, plants and pathogenic bacteria, are similar. All reveal a bent cavity passing close to the non-heme iron involved in catalysis. However, specificity for oxidation at one of several sites on the unsaturated fatty acid substrates lacks an overarching rationale. We have positioned spin labels at selected sites on helices-2, −9, −13, −15 and −22 in soybean lipoxygenase-1 and examined whether the substrate analog lysooleoyllecithin induces conformational changes at any of these sites. Site-specific spin labeling of introduced cysteines was achieved on a cysteine-free background in which each natural cysteine was substituted by serine. EPR spectra revealed conformational changes in helix-2 with lysolecithin binding and with pH variation, while the other sites did not. Site-directed spin labeling of helix-2 includes a spin-label scan of single sites for responses to lyso lecithin and also changes of charged residues in helix-2 to alanines to characterize the pH dependent changes. The spin label sites on the other helices are conformationally invariant and serve as the basis for ongoing of a lipoxygenase structure by paramagnetic distance geometry. All spin labeled lipoxygenase sites examined have enzymatic properties similar to native enzyme.

Rigid Core and Flexible Terminus: STRUCTURE OF SOLUBILIZED LIGHT-HARVESTING CHLOROPHYLL a/b COMPLEX (LHCII) MEASURED BY EPR

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations.