TOAC Spin Label Research Articles

Determining the helical tilt angle and dynamic properties of the transmembrane domains of pinholin S2168 using mechanical alignment EPR spectroscopy

The lytic cycle of bacteriophage φ21 for the infected E. coli is initiated by pinholin S21, which determines the timing of host cell lysis through the function of pinholin (S2168) and antipinholin (S2171). The activity of pinholin or antipinholin directly depends on the function of two transmembrane domains (TMDs) within the membrane. For active pinholin, TMD1 externalizes and lies on the surface while TMD2 remains incorporated inside the membrane forming the lining of the small pinhole. In this study, spin labeled pinholin TMDs were incorporated separately into mechanically aligned POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) lipid bilayers and investigated with electron paramagnetic resonance (EPR) spectroscopy to determine the topology of both TMD1 and TMD2 with respect to the lipid bilayer; the TOAC (2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid) spin label was used here because it attaches to the backbone of a peptide and is very rigid. TMD2 was found to be nearly colinear with the bilayer normal (n) with a helical tilt angle of 16 ± 4° while TMD1 lies on or near the surface with a helical tilt angle of 84 ± 4°. The order parameters (~0.6 for both TMDs) obtained from our alignment study were reasonable, which indicates the samples incorporated inside the membrane were well aligned with respect to the magnetic field (B0). The data obtained from this study supports previous findings on pinholin: TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S2168. In this study, the helical tilt angle of TMD1 was measured for the first time. For TMD2 our experimental data corroborates the findings of the previously reported helical tilt angle by the Ulrich group.

Probing the E/K Peptide Coiled-Coil Assembly by Double Electron-Electron Resonance and Circular Dichroism.

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

Evaluation of 4-tert-Butyl-Benzhydrylamine Resin (BUBHAR) as an Alternative Solid Support for Peptide Synthesis

Following preliminary reports that introduced 4-tert-butylbenzhydrylamine resin (BUBHAR) as a novel polymer for use in solid-phase peptide chemistry (SPPS), some physical-chemical properties of its structure, certainly relevant for its application in this methodology, were compared with those of the largely used methylbenzhydrylamine resin (MBHAR). In order to rule out possible MBHAR-related commercial source effects for SPPS, we initially compared MBHAR batches acquired from three different manufacturers with homemade BUBHARs. The bead solvation properties of these two resins in solvents used in the tert-butyl (Boc-based) SPPS technique indicated that the mean swelling values of these solid supports (% volume of solvated bead occupied by the solvent) were 51% and 67% for MBHAR and BUBHAR, respectively. This result strongly suggests a good potential for the latter polymer in terms of application for application in SPPS. In order to move forward with this approach, the synthesis of the carboxy-terminal peptide fragment (Gln-Asn-Cys-Pro-(D-Arg)-Gly-amide) of the antidiuretic hormone, desmopressin ([3-Mpa∗-Tyr-Phe-Gln-Asn-Cys-Pro-(D-Arg)-Gly-amide], ∗1-[3-mercaptopropionic acid]), which our laboratory is producing routinely in large scale for the Health Secretary of Sao Paulo State. The comparative synthesis was conducted using these two resins with similar substitution degrees (~0.7 mmol/g). In contrast to MBHAR, surprisingly no need for a Gln⟶Asn recoupling reaction was observed when BUBHAR was used. This result might be due to improved solvation of the desmopressin C-terminal Asn-Cys-Pro-(D-Arg)-Gly-segment when bound to this latter resin as observed by microscopic swelling degrees of peptide-resin beads and also by greater mobility detected of peptide chains within the BUBHAR polymer backbone. This finding was determined by comparative electron paramagnetic resonance (EPR) of both peptide resins attaching the amino acid-type paramagnetic 2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (Toac) spin label early introduced by our group.

Assessing topology and surface orientation of an antimicrobial peptide magainin 2 using mechanically aligned bilayers and electron paramagnetic resonance spectroscopy

Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label. The results revealed the helical tilts to be 66° ± 5°, 76° ± 5°, 70° ± 5°, and 72° ± 5° for the TOAC substitutions H7, S8, A9, and K10 respectively. These results are consistent with previously published literature. Using the EPR (electron paramagnetic resonance) mechanical alignment technique, these substitutions were used to critically assess the topology and surface orientation of the peptide with respect to the membrane. This methodology offers a rapid and simple approach to investigate the structural topology of antimicrobial peptides.

EPR Structural Dynamics of Calmodulin-RyR Peptide Complex

We have used a combination of site-directed spin labeling and EPR spectroscopy to probe the interaction of calmodulin (CaM) with a peptide (RyRp) corresponding to the CaM-binding site on the ryanodine receptor, the muscle calcium release channel. A TOAC spin label, which is bifunctionally and stereospecifically coupled to the alpha-carbon and directly reports peptide backbone dynamics, was used to label RyRp at positions 5, 12, 18, or 25. CW-EPR showed that RyRp alone exhibits a gradient of rotational dynamics (mobility), with the N-terminal region having greater mobility than the C-terminal region. Addition of CaM restricted mobility at all four positions, while preserving the mobility gradient. In the presence of saturating Ca, the immobilization of TOAC-RyRp by CaM is further increased. A bifunctional spin label (BSL) attached stereospecifically at Cys sites i and i+4 was used to label CaM at positions 34 and 38 in the N-lobe and 106 and 110 in the C-lobe. Interspin distances measured by DEER indicated that CaM, in the absence of peptide and Ca, exists in a closed conformation, while Ca induces a shift in equilibrium toward two additional conformations, one more open and another more closed (“compact”). Addition of RyRp caused the two lobes of CaM to collapse toward each other (compact conformation), while exhibiting a broad distribution, indicating high flexibility. Removal of Ca from the complex caused increased distance between the spin labels, indicating greater lobe separation. Distances measured between BSL (on CaM) and TOAC (on RyRp) confirmed the compact but flexible conformation of the complex. These results provide insight into the structural dynamics of the Ca-dependent regulation of RyR by CaM.

TOAC Spin Label Conformation Resolved by MD Simulation of Membrane-Bound Phospholamban

We have used all-atom molecular dynamics (MD) simulation to determine the conformational occupancy of the nitroxide spin label TOAC attached to membrane-bound phospholamban (PLB), as needed for interpretation of EPR spectra. EPR spectroscopy is sensitive primarily to the dynamic angular distribution of the probe principal axis relative to the magnetic field, θHP. MD simulations determine directly the orientation of the probe principal axis relative to the α-helix axis, θMP. Previously, θMP has been determined from X-ray crystallography of TOAC on α-helical peptides, which have indicated sole occupancy of a twistboat conformation, with θMP = 13.3° (Marsh 2006 JMagRes). We have employed all-atom MD simulations to determine the distribution of θMP values for TOAC bound to PLB. The PLB structure (PDB ID: 2KB7) was mutated with TOAC at residues 11, 24, 36, or 46, and placed in Monte-Carlo-generated lipid bilayers composed of either DOPC or DMPC:POPC. MD simulations were performed using NAMD with the CHARMM36 force field. Trajectories were computed to 100ns for chair, boat, and twistboat starting conformations of TOAC. Time-averaged geometry of PLB α-helices in the lipid bilayer membrane were calculated, and results agreed with published NMR data, validating the MD results. Furthermore, angle distributions in the probe frame - (θHP, ϕHP) and (θMP, ϕMP) - were calculated, indicating a bimodal TOAC occupancy model with sensitivity to label location. Coupling MD-derived conformational models with EPR data fitting has improved determination of the conformational distribution of PLB in a lipid bilayer and will allow us to determine PLB orientations in other conditions such as when bound to SERCA.

EPR with Rigidly Bound Spin Labels used to Probe the Interaction of Calmodulin with the Ryanodine Receptor

We have used site-directed spin labeling and EPR spectroscopy to probe the structural dynamics of calmodulin bound to a ryanodine receptor target peptide. Calmodulin binds to a conserved 30-amino acid sequence on the sarcoplasmic reticulum calcium release channel (ryanodine receptor, RyR) and directly modulate its activity in muscle contraction. In order to characterize the structural details of this crucial interaction, a bifunctional spin label was used to rigidly label di-cysteine mutant calmodulin at positions 34 and 38. Solid-phase peptide synthesis was used to create a peptide corresponding to the calmodulin binding site on the ryanodine receptor channel (RyRp, residues 3614-3643 in RyR1) with a TOAC spin label rigidly coupled to the backbone. The intermolecular distance distribution between the two spin probes was determined by EPR, using both continuous wave dipolar broadening and dipolar electron-electron resonance (DEER). The center of the distance distribution was in good agreement with the crystal structure of the complex (18 A), but the width of the distribution was ∼ 1 nm, comparable to that observed previously within calmodulin itself. Thus calmodulin retains significant inter-lobe domain motion, even after RyRp binding. These results are consistent with a model in which calmodulin binds RyRp, bringing its two terminal lobes close together but preventing them from physical contact with each other, allowing flexibility in each of the lobe.

CW dipolar broadening EPR spectroscopy and mechanically aligned bilayers used to measure distance and relative orientation between two TOAC spin labels on an antimicrobial peptide

An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3Å from a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3Å distance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.

Conformation and EPR characterization of rigid, 310‐helical peptides with TOAC spin labels: Models for short distances

For 3D-structure determination in biophysical systems EPR is rapidly gaining ground. Proteins labeled specifically with two nitroxide spin labels can be prepared, and several EPR methods are available for distance determination, which makes it possible to determine distance constraints. However, such methods require frozen solutions, potentially causing non-physiological states of the sample. Here, we target spin- spin interaction in liquid solution at room temperature using rigid model compounds. A series of 310 -helical peptides, based on α-aminoisobutyric acid (Aib), is synthesized with pairs of spin labels separated by three, four, and five amino acids. To avoid flexibility, the noncoded nitroxyl-containing α-amino acid TOAC that is rigidly connected with the peptide backbone, is used. The EPR spectra of the peptides show a decreasing amount of coupling between the two spin labels within this series. We suggest through-bond interaction as the dominating mechanism for exchange interaction (J) and find a stronger J-coupling than in the corresponding Ala-based TOAC-peptides investigated previously (Hanson, et al., J Am Chem Soc 1996, 118, 7618-7625). We speculate that stronger coupling in Aib- vs Ala- peptides is due to intrinsically stronger through-bond interaction in the Aib-based peptides.

Incorporation of a Rigid TOAC Spin-Label as a Non-Native Amino Acid into a Full-Length Protein by In Vitro Translation using Amber Codon Suppression

The development of EPR site-directed spin-labeling (SDSL) techniques has provided a powerful avenue for the study of membrane protein structure and dynamics. One of the advantages of EPR is the ability to obtain distance and dynamic information about particular protein regions. The accuracy of this information is highly dependent on the spin-label that is used. Labels with less inherent mobility are better reporters of protein backbone dynamics and give more sharply defined distance parameters. TOAC is a commonly used rigid spin-label that can be integrated into the protein backbone as an amino acid analog. However, its current use is limited to the study of small peptides that can be only generated synthetically (solid-phase peptide synthesis). A new biochemical approach for incorporation of non-natural amino acids during translation using a synthetically loaded amber suppressor tRNA has successfully generated TOAC-labeled protein but not in sufficient yields for EPR studies. Here, we describe the use of an amber codon suppressor tRNA and a continuous exchange cell-free (CECF) expression system for in vitro translation of a small thylakoid membrane protein, Tha4, containing site-specific integration of TOAC. Further, we demonstrate successful incorporation of the TOAC-containing Tha4 into liposomes by supplying POPC vesicles during the translation reaction. By combining new techniques in suppressor tRNA and continuous exchange cell-free (CECF) translation, we have developed a method for translation of full length proteins with site-directed insertion of non-natural amino acid spin labels in yields applicable to CW-EPR experiments.

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy

Wild-type phospholamban (WT-PLB), a Ca2+-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca2+-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100ns rotational correlation time (τc); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.

Molecular Dynamics Simulation of a Spin-Labeled Membrane Protein

We have developed a computational molecular dynamics technique to simulate the motion of the TOAC spin label bound to the cytosolic and transmembrane domains of monomeric phospholamban (PLB) in a lipid bilayer. In cardiac muscle, the integral membrane protein PLB binds to and inhibits the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA), decreasing calcium sequestration into the SR lumen. Previous electron paramagnetic resonance (EPR) experiments showed strong immobilization of the spin probe when placed near the C-terminus of PLB, indicating a stable and highly ordered transmembrane helix. It was shown that the cytosolic domain samples two distinct conformations, an ordered T state with moderately restricted motion and a dynamically disordered R state with nearly unrestricted isotropic motion. Phospholamban regulatory function requires an order-to-disorder transition in the cytosolic domain, with phosphorylation at Ser16 shifting the equilibrium toward the mobile R state and relieving SERCA inhibition. In the present study, monomeric PLB was simulated in a POPC lipid bilayer with the newly parameterized TOAC spin label replacing amino acids at positions 11 and 36. Unlike cysteine-reactive spin labels, TOAC directly reports the motion of the peptide backbone, making it possible to computationally analyze the coupled motion of the transmembrane helix. The simulation for each mutant is performed with and without phosphorylation of Ser16. We optimized molecular dynamics simulation conditions and the resulting trajectories are used to calculate order parameters, orientational distributions, rotational correlation times, and helical tilt angles for comparison with previous experimental results. The dynamics of the spin-labeled protein are compared with the wild type monomer to investigate the effect of the spin probe on the peptide backbone. This computational method informs our current experimental model of TOAC-protein dynamics and strengthens our molecular dynamics modeling technique for performing future spin probe experiments in silico.

Alignment Studies Employing the Rigid TOAC Spin Label Utilizing Electron Paramagnetic Resonance (EPR)

For electron paramagnetic resonance (EPR) spectroscopic studies, the TOAC spin label offers the unique advantage over other conventional labels in that it reports accurate backbone motion and peptide dynamics due to its rigid nature. This label has become extremely important in EPR studies to study membrane protein topology and their associated dynamics. Additionally, some researches have also developed spectroscopic techniques using magnetically aligned (bicelles) and mechanically aligned (glass plates) lipid samples to extract additional information directly related to structural topology with respect to the membrane.

The Temperature Dependence of Nitroxide Spin–Label Interaction Parameters: a High-Field EPR Study of Intramolecular Motional Contributions

High-field W-band electron paramagnetic resonance (EPR) spectroscopy was utilized to study the temperature dependence of the magnetic interaction parameters (g-, hyperfine-, quadrupole tensors) of two types of doublet-state nitroxide spin probes in glass-forming ortho-terphenyl solution: a five-membered ring system of pyrroline type (model for the commonly used methane thiosulfonate spin label) and a six-membered ring system of piperidine type (model for the commonly used TOAC spin label). The analysis of the g- and hyperfine tensors in terms of their isotropic and anisotropic parts reveals at least two mechanisms of motion that are responsible for the temperature dependence of the interaction parameters. The first mechanism is attributed to the overall small-angle motion of the nitroxide molecule in the glassy matrix; it leads to an averaging of the anisotropies of the EPR parameters. The second mechanism originates in an intramolecular out-of-plane motion of oxygen in the nitroxide group. This type of motion is evidenced by comparing the experimental findings for the spin-interaction parameters with the results of density functional theory calculations. The harmonic oxygen out-of-plane vibrations result in a variation of both the isotropic and anisotropic parts of the g- and hyperfine tensors. In contrast, the quadrupole tensor is not influenced by this vibration mechanism in the temperature range under study (90–240 K). Consequences of the applicability of such typical nitroxide radicals for probing details of their protein environment and for studying librational dynamics in frozen solutions are discussed.

Alamethicin Topology in Phospholipid Membranes by Oriented Solid-state NMR and EPR Spectroscopies: a Comparison

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

Spin Label Spin Label Distance Measurements of Aligned Membrane Proteins

Both Manganin-2 and M2δ transmembrane segment of the nicotinic acetylcholine receptor (AchR M2δ) are 23-amino acid peptides with strikingly different structural topologies. Manganin-2 is an antimicrobial peptide lying on the surface, whereas AchR M2δ forms a membrane-spanning neurotransmitter gated ion-channel. To determine the structural topology of these peptides, the spin label 2, 2, 6, 6-tetramethyl piperidine-1 oxyl-4-amino-4 carboxylic acid (TOAC) was attached at different sites in these peptides via solid phase peptide synthesis. Since the TOAC spin label is rigidly coupled to the peptide backbone, it accurately reports on position, orientation, and dynamics of the peptide backbone. Amino acids Ser-8 and Lys-14 in Manganin-2 and Ile-7 and Gln-13 in AchR M2δ were singly and doubly replaced with the TOAC spin label. These peptides were cleaved, purified by reverse phase HPLC and then inserted into aligned DMPC lipid bilayers to study the spin label-spin label dipolar interaction by continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. In aligned bilayers at higher temperatures, the anisotropic EPR spectra of the singly labeled peptides reveal unique lineshapes based upon the orientation of the peptides with respect to the membrane and the magnetic field. The EPR spectra of doubly labeled peptides in aligned bilayers contain unique information about the distance and orientation of the two labels. Thus, by using aligned CW-EPR spectroscopic techniques we can predict the structural topology of membrane proteins and measure the distance and orientation between two spin labels.

Determining the helical tilt of membrane peptides using electron paramagnetic resonance spectroscopy

Theoretical calculations of hyperfine splitting values derived from the EPR spectra of TOAC spin-labeled rigid aligned α-helical membrane peptides reveal a unique periodic variation. In the absence of helical motion, a plot of the corresponding hyperfine splitting values as a function of residue number results in a sinusoidal curve that depends on the helical tilt angle that the peptide makes with respect to the magnetic field. Motion about the long helical axis reduces the amplitude of the curve and averages out the corresponding hyperfine splitting values. The corresponding spectra can be used to determine the director axis tilt angle from the TOAC spin label, which can be used to calculate the helical tilt angle due to the rigidity of the TOAC spin label. Additionally, this paper describes a method to experimentally determine this helical tilt angle from the hyperfine splitting values of three consecutive residues.

Rotational dynamics of HIV-1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis.

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label was attached at the N-terminal position to interrogate the dynamics of the HIV-1 nucleocapsid Zn-finger protein, NCp7. NCp7 is a 6.4-kDa 55-mer critical to the recognition, packaging, and efficient reverse transcription of viral RNA that has stem-loop structures, such as the RNA stem-loop 3 used in this work. The NCp7, made by solid-phase peptide synthesis with TOAC incorporated into the alpha-carbon backbone at the N-terminal "0" position, showed analytical purity and biological activity. Electron Paramagnetic Resonance (EPR) spectra of the N-terminal TOAC indicated rapid temperature-sensitive motion of the probe (< or =0.33 ns correlation time) on the flexible N-terminal segment. This N-terminal TOAC-NCp7 reported a RNA-NCp7 interaction at a 1:1 ratio of NCp7 to RNA, which caused the tumbling time to be slowed from about 0.3 ns to about 0.5 ns. NCp7 is a largely disordered protein that adapts to its RNA targets. However, as shown by circular dichroism, > or =90% trifluoroethanol [(TFE), an alpha-helix enhancer] caused the TOAC-NCp7 without zinc in its fingers to change to a fully helical conformation, while the TOAC spin label was concurrently reporting a tumbling time of well over a nanosecond, as the N-terminal TOAC became inflexibly enfolded. Even with TFE present, the existence of intact Zn-finger regions in NCp7 prevented complete formation of helical structure, as shown by circular dichroism, and decreased the N-terminal TOAC tumbling time, as shown by EPR. This study demonstrated TOAC at the N-terminal of NCp7 to be a reporter for the considerable conformational lability of NCp7. (

Rotational Dynamics of Phospholamban Determined by Multifrequency Electron Paramagnetic Resonance

We have used multifrequency electron paramagnetic resonance to define the multistate structural dynamics of an integral membrane protein, phospholamban (PLB), in a lipid bilayer. PLB is a key regulator of cardiac calcium transport, and its function requires transitions between distinct states of intramolecular dynamics. Monomeric PLB was synthesized with the TOAC spin label at positions 11 (in the cytoplasmic domain) and 46 (in the transmembrane domain) and reconstituted into lipid bilayers. Unlike other protein spin labels, TOAC reports directly the motion of the peptide backbone, so quantitative analysis of its dynamics is worthwhile. Electron paramagnetic resonance spectra at 9.4 GHz (X-band) and 94 GHz (W-band) were analyzed in terms of anisotropic rotational diffusion of the two domains. Motion of the transmembrane domain is highly restricted, while the cytoplasmic domain exhibits two distinct conformations, a major one with moderately restricted nanosecond dynamics ( T) and another with nearly unrestricted subnanosecond motion ( R). The global analysis of spectra at two frequencies yielded values for the rotational correlation times and order parameters that were much more precisely determined than at either frequency alone. Multifrequency EPR is a powerful approach for analysis of complex rotational dynamics of proteins.

Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label at the N-terminal (TOAC1-AngII and TOAC0-BK) and internal (TOAC3-AngII and TOAC3-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT1 and B2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. In contrast to internally labeled analogues (TOAC3-AngII or TOAC3-BK), the TOAC1-AngII and TOAC0-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT1 or B2 receptors, respectively. In addition, TOAC1-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC0-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC3-AngII or TOAC3-BK did not provoke any alteration in blood pressure levels. In summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important tool to evaluate peptide–receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT1 and B2 receptors.