Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin‐Labeling and EPR Spectroscopy

Drug binding to human serum albumin (HSA) has been characterized by a spin‐labeling and continuous‐wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin‐binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR‐active nitroxide radicals (spin‐labeled pharmaceuticals (SLPs)) and in a screening approach CW‐EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW‐EPR spectra allow extraction of association constants (K_A) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug–protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged.     

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.     

Properties of polysaccharides grafted on nanoparticles investigated by EPR

The in vivo fate of nanoparticles developed as drug delivery systems is influenced by the surface characteristics of the colloidal particles. In the present work, surface characteristics of a series of poly(isobutylcyanoacrylate) nanoparticles prepared by redox radical emulsion polymerization with polysaccharides of different molecular weight and nature were characterized by EPR. To this aim, a spin label was grafted on the polysaccharide chains after synthesis of the nanoparticles. The percentage of label showing fast movements was evaluated from EPR spectra which were analyzed according to the Kivelson theory. The results showed that mobility depended on temperature, type, and molecular weight of the polysaccharides. Differences between nanoparticles appeared with low-molecular-weight polysaccharides, while over a defined molecular weight which depended on the nature of the polysaccharide, the spin label behaved almost the same way in the different types of nanoparticles. Paradoxically, the percentage of fast moving label was the highest when linked to the shortest chitosan, which was the most rigid polysaccharide tested in this study. Thus, it was concluded that the apparent mobility of the polysaccharide evaluated by the EPR method depended on the capacity of the polysaccharide chains to fold making possible hydrophobic interactions between the label and the nanoparticle core. The transition between the unfolded-folded regiment depended on the molecular weight and on the nature of the polysaccharide. Results of this study may be useful to improve the understanding of the nanoparticle interactions with blood proteins and complement which in turn influence the in vivo fate of nanoparticles used as drug delivery systems.     

Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time T_M for HSA labeled with OX063 is 6.3 μs at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.     

Calculating slow-motional electron paramagnetic resonance spectra from molecular dynamics using a diffusion operator approach

A number of groups have utilized molecular dynamics (MD) to calculate slow-motional electron paramagnetic resonance (EPR) spectra of spin labels attached to biomolecules. Nearly all such calculations have been based on some variant of the trajectory method introduced by Robinson, Slutsky and Auteri (J. Chem. Phys. 1992,96, 2609-2616). Here we present an alternative approach that is specifically adapted to the diffusion operator-based stochastic Liouville equation (SLE) formalism that is also widely used to calculate slow-motional EPR line shapes. Specifically, the method utilizes MD trajectories to derive diffusion parameters such as the rotational diffusion tensor, diffusion tilt angles, and expansion coefficients of the orienting potential, which are then used as direct inputs to the SLE line shape program. This approach leads to a considerable improvement in computational efficiency over trajectory-based methods, particularly for high frequency, high field EPR. It also provides a basis for deconvoluting the effects of local spin label motion and overall motion of the labeled molecule or domain: once the local motion has been characterized by this approach, the label diffusion parameters may be used in conjunction with line shape analysis at lower EPR frequencies to characterize global motions. The method is validated by comparison of the MD predicted line shapes to experimental high frequency (250 GHz) EPR spectra.     

Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.     

Post‐synthetic Spin‐Labeling of RNA through Click Chemistry for PELDOR Measurements

Site‐directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron‐electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here d? . We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW‐EPR as well as PELDOR we analyzed and compared the influence of d? and Çm on a self‐complementary RNA duplex. Our results demonstrate that the conformational diversity of d? is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation‐selective PELDOR time traces of the d? ‐labeled RNA duplex.     

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for ¹H and ¹³C (¹H→¹³C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for ¹H and 13.9±0.5 for ¹³C (direct polarization).     

High-field EPR and ESEEM investigation of the nitrogen quadrupole interaction of nitroxide spin labels in disordered solids: toward differentiation between polarity and proticity matrix effects on protein function

The combination of high-field electron paramagnetic resonance (EPR) with site-directed spin labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in revealing subtle changes of the polarity and proticity profiles in proteins enbedded in membranes. This information can be obtained by orientation-selective high-field EPR resolving principal components of the nitroxide Zeeman (g) and hyperfine ( A) tensors of the spin labels attached to specific molecular sites. In contrast to the g- and A-tensors, the (14)N ( I = 1) quadrupole interaction tensor of the nitroxide spin label has not been exploited in EPR for probing effects of the microenvironment of functional protein sites. In this work it is shown that the W-band (95 GHz) high-field electron spin echo envelope modulation (ESEEM) method is well suited for determining with high accuracy the (14)N quadrupole tensor principal components of a nitroxide spin label in disordered frozen solution. By W-band ESEEM the quadrupole components of a five-ring pyrroline-type nitroxide radical in glassy ortho-terphenyl and glycerol solutions have been determined. This radical is the headgroup of the MTS spin label widely used in SDSL protein studies. By DFT calulations and W-band ESEEM experiments it is demonstrated that the Q(yy) value is especially sensitive to the proticity and polarity of the nitroxide environment in H-bonding and nonbonding situations. The quadrupole tensor is shown to be rather insensitive to structural variations of the nitroxide label itself. When using Q(yy) as a testing probe of the environment, its ruggedness toward temperature changes represents an important advantage over the g xx and A(zz) parameters which are usually employed for probing matrix effects on the spin labeled molecular site. Thus, beyond measurenments of g xx and A(zz) of spin labeled protein sites in disordered solids, W-band high-field ESEEM studies of (14)N quadrupole interactions open a new avenue to reliably probe subtle environmental effects on the electronic structure. This is a significant step forward on the way to differentiate between effects from matrix polarity and hydrogen-bond formation.     

Phase Separation of Poly(N‐isopropylacrylamide) in Mixtures of Water and Methanol: A Spectroscopic Study of the Phase‐Transition Process with a Polymer Tagged with a Fluorescent Dye and a Spin Label

The thermoreversible phase transition of poly(N‐isopropylacrylamide) randomly labeled with a spin label, 4‐amino‐2,2′,6,6′‐tetramethylpiperidine 1‐oxide (TEMPO), and a fluorescent dye, 4‐(pyren‐1‐yl)butyl (PNIPAM‐Py‐T), in different H₂O/MeOH mixtures was studied by turbidimetry, continuous‐wave electron paramagnetic resonance spectroscopy (CW‐EPR), and fluorescence spectroscopy. The macroscopic phase diagram of PNIPAM‐Py‐T in H₂O/MeOH measured by turbidimetry was identical to those of poly(N‐isopropylacrylamide) (PNIPAM) and of TEMPO‐labeled PNIPAM (PNIPAM‐T) in H₂O/MeOH mixtures. However, distinct differences among the three polymers were detected in their solvent‐dependent EPR and fluorescence‐spectroscopic properties. The EPR spectra were analyzed in terms of the isotropic hyperfine coupling constants, which monitor the variation in environmental polarity of the radical labels occurring for the conformational transitions of the polymer as a function of temperature, as well as the correlation time for reorientation motion, the increase of which is indicative of the increased viscosity of the radical environment and interactions occurring between the radical and other surface groups of the precipitated polymer, if compared to the soluble polymer. The fluorescence of Py in PNIPAM‐Py‐T displayed contributions from isolated excited pyrenes (monomer emission) and from preformed pyrene ground‐state aggregates (excimer emission). The quantum efficiencies of monomer and excimer emission were monitored as a function of solvent composition. By the two experimental approaches, we demonstrate the profound influence of the PNIPAM‐attached pyrene units in increasing the hydrophobicity of the nanodomains formed upon heat‐induced precipitation of PNIPAM‐Py‐T.     

SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site‐directed spin labeling experiments

Site‐directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the residue level, structural transitions in proteins. SDSL‐EPR is based on the selective grafting of a paramagnetic label on the protein under study, followed by cw EPR analysis. To extract valuable quantitative information from SDSL‐EPR spectra and thus give reliable interpretation on biological system dynamics, numerical simulations of the spectra are required. Such spectral simulations can be carried out by coding in MATLAB using functions from the EasySpin toolbox. For non‐expert users of MATLAB, this could be a complex task or even impede the use of such simulation tool. We developed a graphical user interface called SimLabel dedicated to run cw EPR spectra simulations particularly coming from SDSL‐EPR experiments. Simlabel provides an intuitive way to visualize, simulate, and fit such cw EPR spectra. An example of SDSL‐EPR spectra simulation concerning the study of an intrinsically disordered region undergoing a local induced folding is described and discussed. We believe that this new tool will help the users to rapidly obtain reliable simulated spectra and hence facilitate the interpretation of their results. Copyright © 2017 John Wiley & Sons, Ltd.     

Small-angle X-ray scattering and electron paramagnetic resonance study of the interaction of bovine serum albumin with ionic surfactants

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) techniques have been used to monitor the interaction of bovine serum albumin (BSA) with ionic surfactants such as anionic sodium dodecyl sulfate (SDS), zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate (HPS), and cationic cethyltrimethylammonium chloride (CTAC) at pH 7.0. The SAXS results have shown that in the presence of 5 mM SDS and HPS the radius of gyration (Rg) almost does not change as compared to the BSA free-surfactant solution; its value is ca. 30 Angstroms. In the presence of 5 mM CTAC the SAXS data indicate the presence of a particle with a Rg of at least 63 Angstroms, suggesting that in this case, a kind of protein aggregation takes place. In the presence of SDS and HPS surfactants at concentrations above 10 mM, a characteristic broad peak in the region of 0.12-0.18 Angstroms(-1) indicates the presence of micelle-like aggregates in solution. The SAXS curves are consistent with the "pearl necklace" model, where micelle-like aggregates are randomly distributed around the polypeptide chain. EPR results using 5-DSA and 16-DSA spin labels show that in the presence of BSA the EPR spectra are composed of two label populations, one contacting the protein and a second one due to label localization in the micelles. Evidence is also obtained for a competition of the surfactants with the spin labels for the high-affinity binding sites of the stearic acid spin labels as monitored by changes in the fractions of the two label populations as the surfactant concentration is increased. The effect of SDS seems to be stronger in the sense that increased SDS concentration leads to a complete transfer of spin labels from close protein contact sites to micelles, while for HPS, a significant immobilization of probe apparently remains even at higher surfactant concentrations. These two techniques are quite useful since SAXS monitors the overall properties of the scattering particle, while EPR gives information on the dynamics inside this particle and associated with label localization and motion.     

Mapping electron paramagnetic resonance spin label conformations by the simulated scaling method

In order to efficiently simulate spin label behavior when attached to the protein backbone we developed a novel approach that enhances local conformational sampling. The simulated scaling (SS) approach (Li, H., et al. J. Chem. Phys. 2007, 126, 24106) couples the random walk of a potential scaling parameter and molecular dynamics in the framework of hybrid Monte Carlo. This approach allows efficient barrier crossings between conformations. The method retains the thermodynamic detailed balance allowing for determination of relative free energies between various conformations. The accuracy of our method was validated by comparison with the recently resolved X-ray crystal structure of a spin labeled T4 lysozyme in which the spin label was in the interior of the protein. Consistent potentials of mean force (PMF) are obtained for the spin label torsion angles to illustrate their behavior in various protein environments: surface, semiburied, and buried. These PMFs reflect the experimentally observed trends and provide the rationale for the spin label dynamics. We have used this method to compare an implicit and explicit solvent model in spin label modeling. The implicit model, which is computationally faster, was found to be in excellent agreement with the explicit solvent treatment. Based on this collection of results, we believe that the presented approach has great potential in the general strategy of describing the behavior of the spin label using molecular modeling and using this information in the interpretation of EPR measurements in terms of protein conformation and dynamics.     

Interaction of tetraphenyl-porphyrin derivatives with DPPC-liposomes: an EPR study

The effect of the symmetry and polarity of the porphyrin molecules on their membrane localization and interaction with membrane lipids were investigated by electron paramagnetic resonance (EPR). For this purpose, two glycoconjugated tetraphenyl porphyrin derivatives were selected, respectively, symmetrically and asymmetrically substituted. Small unilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and spin labeled stearic acids were prepared. The spin probe was located at the 5th or 7th or 12th or 16th position of the hydrocarbon chain in order to monitor various regions of the lipid bilayer. EPR spectra of porphyrin-free and porphyrin-bound liposomes were recorded at various temperatures below and above the phase transition temperature of DPPC. The effect on membrane fluidity proved to be stronger with the asymmetrical porphyrin derivative than with the symmetrical one. The rigidity increased when the spin label was near lipid head groups. The difference observed between control and porphyrin-treated samples when measured below the main lipid transition temperature disappeared at higher temperature. When the spin label was near the end of the hydrophobic tails, the symmetrical porphyrin derivative caused increase in fluidity, while the asymmetrical one slightly decreased it. To explain this phenomenon we propose that the asymmetrical derivative exerts a stronger ordering effect caused by its fluorophenyl group located at the level of the lipid heads, which is attenuated to the hydrophobic tails. The perturbing effect of the symmetric derivative could not lead to similar extent of ordering at the head groups and looses the hydrocarbon chains deeper in the membrane.     

A Bioresistant Nitroxide Spin Label for In‐Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies

Approaching protein structural dynamics and protein–protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site‐directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide‐based spin labels for in‐cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido‐proxyl‐based spin label (M‐TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M‐TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.     

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.     

A Spirocyclohexyl Nitroxide Amino Acid Spin Label for Pulsed EPR Spectroscopy Distance Measurements

Site‐directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide α‐amino acid and its N‐(9‐fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin‐echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR spectroscopy to the study of structure and conformation of peptides and proteins.     

Selective binding of mannose-encapsulated gold nanoparticles to type 1 pili in Escherichia coli

The synthesis, characterization and biological application of mannose encapsulated gold nanoparticles (m-AuNP) are reported. m-AuNP is well dispersed and very stable without aggregation in the media of broad ion strength and pH ranges. The selective binding of m-AuNP to the mannose adhesin FimH of bacterial type 1 pili is demonstrated using transmission electron microscopy. The competition assay with free mannose suggests that m-AuNP binds FimH better than free mannose does. This work demonstrates that carbohydrate attached nanoparticles can be used as an efficient affinity label and a multi-ligand carrier in a biological system.