Paramagnetic Relaxation Research Articles

Abstract 5238: Structural and biochemical characterization of membrane bound KRAS

Abstract KRAS4b functions as a molecular switch cycling between an active GTP bound state and inactive GDP state. Active KRAS4b binds tightly to RAF1 via its Ras binding domain (RBD), however activation of RAF1 competent for MAPK signal transduction requires membrane bound KRAS4b. Using recombinant farnesylated and methylated KRAS4b (KRAS4b-FMe) we have used a variety of biophysical methodologies including paramagnetic relaxation enhancement NMR, neutron reflectivity, small angle neutron and x-ray scattering and protein footprinting to gain insights into the orientation and structure of KRAS-FMe bound to a membrane. In these experiments we have used Nanodiscs or tethered lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline or -serine (70% POPC 30% POPS). Our data indicate that KRAS-FMe is oriented perpendicular to the membrane with minimal direct contacts to the membrane. The nucleotide bound state does not significantly change the orientation relative to the membrane. We will also present data on the effect of the RBD domain of RAF1 binding to KRAS-FMe at the membrane. Citation Format: Andrew G. Stephen, Que Van, Marco Tonelli, William Gillette, Dominic Esposito, Ben Niu, Michael Gross, Frank Heinrich, Arvind Ramanathan, Christopher Stanley. Structural and biochemical characterization of membrane bound KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2017-5238

Albumin and Hyaluronic Acid-Coated Superparamagnetic Iron Oxide Nanoparticles Loaded with Paclitaxel for Biomedical Applications.

Super paramagnetic iron oxide nanoparticles (SPION) were augmented by both hyaluronic acid (HA) and bovine serum albumin (BSA), each covalently conjugated to dopamine (DA) enabling their anchoring to the SPION. HA and BSA were found to simultaneously serve as stabilizing polymers of Fe3O4·DA-BSA/HA in water. Fe3O4·DA-BSA/HA efficiently entrapped and released the hydrophobic cytotoxic drug paclitaxel (PTX). The relative amount of HA and BSA modulates not only the total solubility but also the paramagnetic relaxation properties of the preparation. The entrapping of PTX did not influence the paramagnetic relaxation properties of Fe3O4·DA-BSA. Thus, by tuning the surface structure and loading, we can tune the theranostic properties of the system.

Gd3+-chelated lipid accelerates solid-state NMR spectroscopy of seven-transmembrane proteins.

Solid-state NMR (SSNMR) is an attractive technique for studying large membrane proteins in membrane-mimetic environments. However, SSNMR experiments often suffer from low efficiency, due to the inherent low sensitivity and the long recycle delays needed to recover the magnetization. Here we demonstrate that the incorporation of a small amount of a Gd3+-chelated lipid, Gd3+-DMPE-DTPA, into proteoliposomes greatly shortens the spin-lattice relaxation time (1H-T 1) of lipid-reconstituted membrane proteins and accelerates the data collection. This effect has been evaluated on a 30kDa, seven-transmembrane protein, Leptosphaeria rhodopsin. With the Gd3+-chelated lipid, we can perform 2D SSNMR experiments 3 times faster than by diamagnetic control. By combining this paramagnetic relaxation-assisted data collection with non-uniform sampling, the 3D experimental times are reduced eightfold with respect to traditional 3D experiments on diamagnetic samples. A comparison between the paramagnetic relaxation enhancement (PRE) effects of Cu2+- and Gd3+-chelated lipids indicates the much higher relaxivity of the latter. Hence, a tenfold lower concentration is needed for Gd3+-chelated lipids to achieve comparable PRE effects to Cu2+-chelated lipids. In addition, Gd3+-chelated lipids neither alter the protein structures nor induce significant line-width broadening of the protein signals. This work is expected to be beneficial for structural and dynamic studies of large membrane proteins by SSNMR.

Search for Electron Delocalization from [Fe(CN)6]3– to the Dication of Viologen in (DNP)3[Fe(CN)6]2·10H2O

K3Fe(CN)6 reacts with the viologen 1,1'-bis(2,4-dinitrophenyl)-4,4'-bipyridinium dication, (DNP)2+, to form a supramolecular complex, (DNP)3[Fe(CN)6]2·10H2O (1). The crystal structure of 1 reveals that there are two [Fe(CN)6]3- anions within an organic framework of three (DNP)2+ cations with the shortest Fe(III)···Fe(III) distances of ca. 9.8 Å, distances that minimize extensive long-range magnetic exchange coupling interactions between the [Fe(CN)6]3- anions, and, thus, 1 is paramagnetic above ca. 17 K and exhibits weak ferromagnetic coupling between 17 and 3 K and antiferromagnetic coupling between 3 and 1.8 K. The long Fe(III)···Fe(III) distances permit slow spin-spin and slow spin-lattice paramagnetic relaxation, relative to the iron-57 Larmor precession frequency, as is evidenced by the Mössbauer spectra measured between 3 and 60 K; between 85 and 295 K, rapid paramagnetic relaxation is observed. Both the slow spin-spin and slow spin-lattice relaxation are mediated by the organic, π-conjugated viologen cations. The Fe-C distances, the Mössbauer isomer shifts, the temperature dependence of the magnetic susceptibility, and the 3 K magnetization results all indicate the presence of low-spin Fe(III) ions in the [Fe(CN)6]3- anions in 1. There is no unequivocal indication of the presence of any formal electron delocalization or transfer from the [Fe(CN)6]3- anion to the (DNP)2+ cations in the results obtained from X-ray crystallography, magnetic measurements, and Mössbauer spectra. Because of enhancement of the spin-orbit coupling by the heavy-atom or -ion effect, the Fe(III) ions in the [Fe(CN)6]3- anions interact with the (DNP)2+ cations, causing them to fluoresce with increasing intensity upon cooling from 90 to 25 K when excited at 300 nm. The resulting luminescence of the viologen (DNP)2+ cation induced by the [Fe(CN)6]3- anions indicates the presence of significant mixing of the molecular orbitals derived from the [Fe(CN)6]3- anions and the molecular orbitals associated with the (DNP)2+ cations to yield bonding supramolecular orbitals in 1, a mixing that is also observed between 50 and 3 K in the temperature dependence of the isomer shift of 1.

Solution Conformations and Dynamics of Substrate-Bound Cytochrome P450 MycG

MycG is a P450 monooxygenase that catalyzes the sequential hydroxylation and epoxidation of mycinamicin IV (M-IV), the last two steps in the biosynthesis of mycinamicin II, a macrolide antibiotic isolated from Micromonospora griseorubida. The crystal structure of MycG with M-IV bound was previously determined but showed the bound substrate in an orientation that did not rationalize the observed regiochemistry of M-IV hydroxylation. Nuclear magnetic resonance paramagnetic relaxation enhancements provided evidence of an orientation of M-IV in the MycG active site more compatible with the observed chemistry, but substrate-induced changes in the enzyme structure were not characterized. We now describe the use of amide 1H-15N residual dipolar couplings as experimental restraints in solvated "soft annealing" molecular dynamics simulations to generate solution structural ensembles of M-IV-bound MycG. Chemical shift perturbations, hydrogen-deuterium exchange, and 15N relaxation behavior provide insight into the dynamic and electronic perturbations in the MycG structure in response to M-IV binding. The solution and crystallographic structures are compared, and the possibility that the crystallographic orientation of bound M-IV represents an inhibitory mode is discussed.

Impact of spin label rigidity on extent and accuracy of distance information from PRE data.

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, UreidoU-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromatic 13C bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Çm is slightly better than the data quality for the flexible UreidoTEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).

Analysis of O2-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A

Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O2-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O2-accessible sites in the protein based on the 1/r6 distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O2 generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O2 within the largest cavity, egress of O2 from the cavity, and ingress of O2 in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O2-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.

Electron Decoupling with Dynamic Nuclear Polarization in Rotating Solids.

Dynamic nuclear polarization (DNP) can enhance NMR sensitivity by orders of magnitude by transferring spin polarization from electron paramagnetic resonance (EPR) to NMR. However, paramagnetic DNP polarizing agents can have deleterious effects on NMR signals. Electron spin decoupling can mitigate these paramagnetic relaxation effects. We demonstrate electron decoupling experiments in conjunction with DNP and magic-angle-spinning NMR spectroscopy. Following a DNP and spin diffusion period, the microwave irradiation frequency is quickly tuned on-resonance with electrons on the DNP polarizing agent. The electron decoupling performance shows a strong dependence on the microwave frequency and DNP polarization time. Microwave frequency sweeps through the EPR line shape are shown as a time domain strategy to significantly improve electron decoupling. For 13C spins on biomolecules frozen in a glassy matrix, electron decoupling reduces the line widths by 11% (47 Hz) and increases the intensity by 14%.

Spherical Polyelectrolyte Brushes as a Novel Platform for Paramagnetic Relaxation Enhancement and Passive Tumor Targeting.

A novel platform for the development of highly efficient magnetic resonance imaging (MRI) contrast agents has been demonstrated. New contrast agents are designed and produced through electrostatic self-assembly of cationic gadolinium(III) complexes onto anionic spherical polyelectrolyte brushes (SPB). The structurally well-defined SPB are composed of polystyrene core and polyacrylic acid brush layer, where numerous binding sites and confined microenvironments are available for the embedment of the gadolinium(III) contrast agents. Both in vitro and in vivo experiments show excellent biocompatibility and relaxometric performance of these SPB-based gadolinium hybrid materials. The enhanced relaxivity value is up to 86.2 mM-1 s-1 per Gd, a remarkably high record value at 1.5 T magnetic field. In vivo imaging displays a prolonged blood circulation time and massive accumulation of the contrast agents at the tumor region due to the enhanced permeability and retention effect. The SPB-based gadolinium hybrid materials not only broaden the horizons of new MRI contrast agents, but also have a great potential for tumor diagnosis.

NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function

Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-1α has remained limited. Here, we report the 3D NMR solution structure of homodimeric CC MBS in which amino acids 932-967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at a and d heptad positions. Using NMR chemical-shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. 15N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation enhancement- and CSP-NMR-guided HADDOCK modeling of the dimer-dimer interface of the heterotetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS·LZ PKG-Iα low-affinity heterotetrameric complex and allow reevaluation of the role(s) of myosin light-chain phosphatase partner polypeptides in regulation of vascular smooth-muscle cell contractility.

Effect of Alignment on Polarized Infrared Emission from Polycyclic Aromatic Hydrocarbons

Abstract Polarized emission from polycyclic aromatic hydrocarbons (PAHs) potentially provides a new way to test the basic physics of the alignment of ultrasmall grains. In this paper, we present a new model of polarized PAH emission that takes into account the effect of PAH alignment with the magnetic field. We first generate a large sample of the grain angular momentum by simulating the alignment of PAHs due to resonance paramagnetic relaxation that accounts for various interaction processes. We then calculate the polarization level of the PAH emission features for the different phases of the interstellar medium, including the cold neutral medium (CNM), reflection nebulae (RNe), and photodissociation regions. We find that a moderate degree of PAH alignment can significantly enhance the polarization degree of the PAH emission compared to the previous results obtained with randomly oriented angular momentum. In particular, we find that the smallest negatively charged PAHs in RNe can be excited to slightly suprathermal rotation due to enhanced ion collisional excitation, resulting in an increase of the polarization with the ionization fraction. Our results suggest that an RN is the most favorable environment in which to observe polarized PAH emission and to test the alignment physics of nanoparticles. Finally, we present an explicit relationship between the polarization level of PAH emission and the degree of external alignment for the CNM and RNe. The obtained relationship will be particularly useful for testing the alignment physics of PAHs in future observations.

Cu(II)-Based Paramagnetic Probe to Study RNA-Protein Interactions by NMR.

Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.

Solvent suppression in DNP enhanced solid state NMR

We show how DNP enhanced solid-state NMR spectra can be dramatically simplified by suppression of solvent signals. This is achieved by (i) exploiting the paramagnetic relaxation enhancement of solvent signals relative to materials substrates, or (ii) by using short cross-polarization contact times to transfer hyperpolarization to only directly bonded carbon-13 nuclei in frozen solutions. The methods are evaluated for organic microcrystals, surfaces and frozen solutions. We show how this allows for the acquisition of high-resolution DNP enhanced proton-proton correlation experiments to measure inter-nuclear proximities in an organic solid.

Human βB2-Crystallin Forms a Face-en-Face Dimer in Solution: An Integrated NMR and SAXS Study

βγ-Crystallins are long-lived eye lens proteins that are crucial for lens transparency and refractive power. Each βγ-crystallin comprises two homologous domains, which are connected by a short linker. γ-Crystallins are monomeric, while β-crystallins crystallize as dimers and multimers. In the crystal, human βB2-crystallin is a domain-swapped dimer while the N-terminally truncated βB1-crystallin forms a face-en-face dimer. Combining and integrating data from multi-angle light scattering, nuclear magnetic resonance, and small-angle X-ray scattering of full-length and terminally truncated human βB2-crystallin in solution, we show that both these βB2-crystallin proteins are dimeric, possess C2 symmetry, and are more compact than domain-swapped dimers. Importantly, no inter-molecular paramagnetic relaxation enhancement effects compatible with domain swapping were detected. Our collective experimental results unambiguously demonstrate that, in solution, human βB2-crystallin is not domain swapped and exhibits a face-en-face dimer structure similar to the crystal structure of truncated βB1-crystallin.

Solid-state NMR and EPR Spectroscopy of Mn2+ -Substituted ATP-Fueled Protein Engines.

Paramagnetic metal ions deliver structural information both in EPR and solid-state NMR experiments, offering a profitable synergetic approach to study bio-macromolecules. We demonstrate the spectral consequences of Mg2+ / Mn2+ substitution and the resulting information contents for two different ATP:Mg2+ -fueled protein engines, a DnaB helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.

Structural Insights into G-tract Recognition by the hnRNP H-RNA Recognition Motif

The heterogeneous nuclear ribonucleoprotein H (hnRNP H) family of proteins are involved in RNA splicing of cellular and viral mRNAs. These proteins function as both splicing activators and repressors. The hnRNP H family proteins have previously been found to interact with poly-G sequences (G-tracts) of cellular and viral mRNAs using quasi RNA recognition motifs (qRRMs) including human immunodeficiency virus (HIV). hnRNP H proteins are composed of three qRRMS, which are separated by linkers and two Glycine rich domains at C-terminal. The qRRM1 and qRRM2 domains are located at the N-terminus and separated by 10-residue linker, whereas qRRM3 is located towards the C-terminus. To gain structural insights into hnRNP H protein, here we solved the solution structure of the HRRM12 domain of hnRNP H using NMR spectroscopy. We used paramagnetic relaxation enhancement (PRE) and Residual dipolar couplings (RDC) to obtain distance restraints and orientational restraints between the HRRM1 and HRRM2 domains. T1, T2 and NOE experimental data is consistent with calculated structure of HRRM12 domain and reveals that HRRM12 adopt the compact (closed) structure. To better understand principals of hnRNP H-RNA recognition, we systematically screened G-tracts of RNA oligos against HRRM12 by using isothermal titration calorimetry (ITC) and NMR spectroscopy. We examined the minimal G-tract RNA (--GGG--) sequence requirements for HRRM12 binding. The HRRM12 domain of hnRNP H exhibited no substantial differences in binding affinities for G-tracts of RNA (AGGGX) and HRRM12 residues involved in binding G-tracts of RNA were detected by NMR chemical shift perturbation experiments and a data-driven model of the complex was determined using HADDOCK. Taken together, this study provides the molecular insights for better understanding the role of the qRRM domains of hnRNP H in RNA splicing of cellular and viral mRNAs.

Conformation Plasticity and Peptidoglycan Cleavage by the N-Terminal Intrinsically Disordered Domain of ChiZ

ChiZ is a transmembrane protein from Mycobacterium tuberculosis involved in the control of cell division. While the exact action of this protein is unknown, ChiZ can indirectly interfere with FtsZ ring formation. Interestingly, the N-terminal cytoplasmic region is able to hydrolyze peptidoglycan (PG) in vitro, even though it is intrinsically disordered. Based on sequence alignment of N-terminus, it is possible to identify a conserved 26 amino acid stretch required for PG hydrolysis and located just before the single transmembrane helix. Although the first 38 residues of the N-terminus are not conserved, a highly positively charged sequence is essential for activity. Using solution NMR tools to characterize the N-terminal region, it is possible to show that the conserved region has different dynamics, proton exchange rates, and tendency to form transient secondary structure compared to the non-conserved region. To assess the relevance of these differences, the N-terminal region was studied in the presence of liposomes and pure PG. The N-terminal region is able to bind liposomes but it is mostly detectable with INEPT based solid state NMR experiments, indicating that this region remains highly dynamic, even in the reconstituted full length protein. Paramagnetic relaxation enhancement experiments showed that the interaction of N-terminal region with lipids is mediated by electrostatic interactions between arginine side chains and lipid head groups. Other residues affected by the paramagnetic ion were located mainly in the middle region. Interestingly, N-terminal binding to PG is similar to binding lipids and it is mediated by arginine and proline rich regions. This data suggests that the loose binding of the N-terminal region to lipids may facilitate the sampling of conformations that can recognize substrate for catalysis. Speculation as to this function in the cytoplasmic domain will be presented.

NMR Studies of Conformational Selection of hnRNP H on RNA Splicing

Modularity provides proteins versatility in regulating many cellular processes. The RNA Recognition Motif (RRM) is a common protein family that adopts modularity to facilitate its biological activity. hnRNP H contains three RRMs that play important roles in RNA splicing by binding G-rich enhancer element2. However, the mechanism three RRMs use to recognize RNA is still unknown. Here, based on the analysis of paramagnetic relaxation enhancement (PRE), the data indicates that hnRNP H RRM12 adopts a closed conformation in the absence of RNA. Second, dynamics experiments, T1, T2 and NOE also reveals that RRM1 and RRM2 domain share similar motion, which suggests that compact (closed) form is major population in solution. Moreover, in closed form, one of two bindings site was buried in compact structure. Based on above observation, we propose a hypothesis that there is chemical equilibrium between open form and close form. Once hnRNP H recognizes its G-rich binding sequencing, it will release the blocking RRM from closed form and reestablish chemical equilibrium toward to open form through conformational selection in presence of RNA3. Integrated methods like NMR paramagnetic relaxation enhancement, dynamics experiments and SAXS would be utilized to address this hypothesis.

Generating a Model for Calmodulin Induced Folding in the Disordered Regulatory Domain of Calcineurin

Calcineurin (CaN) plays an important role in the T-cell activation, cardiac system development and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an α-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal half as well. Here, we are interested in the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13Cα, 13Cβ and HN chemical shifts of the unbound, regulatory domain of CaN. While the protein is disordered, secondary chemical shifts indicate that some regions possess α-helical propensity, even in the unbound state. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 54 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I9-A15. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

Development of highly-sensitive detection system in 19F NMR for bioactive compounds based on the assembly of paramagnetic complexes with fluorinated cubic silsesquioxanes

This manuscript reports the detection system with 19F NMR probes for bioactive amine compounds at sub-micro molar concentrations. Water-soluble fluorinated polyhedral oligomeric silsesquioxane (POSS) derivatives were prepared, and the paramagnetic Ni-porphyrin (NiPor) with the fluorinated POSS (F-POSS) was adsorbed in the buffer. By adding the series of amine derivatives to the homogeneous dispersion containing the POSS-NiPor complexes, the changes in the signal intensity were evaluated. It was clearly observed from homogeneous dispersions that the signal intensity of 19F NMR decreased especially in the presence of ethanediamine and hexylamine. From the dynamic light scattering measurements, it was revealed that the assembly of the F-POSS complexes was dramatically induced by the amine compounds when the signal intensity decreased. According to these data, it is proposed that the signal reduction of 19F NMR from the POSS-NiPor complexes could be induced via the strong paramagnetic relaxation enhancement from NiPor in the assembly. Finally, based on the signal reduction by the assembly, we can detect the amine derivatives under 1μM concentration which is similar sensitivity to the conventional fluorescence probes. Our system is valid to monitor the distribution of the bio-active molecules at cellular concentrations.