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.     

A Bifunctional Spin Label for Ligand Recognition on Surfaces

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin‐labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin‐labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy‐functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin‐labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose‐binding lectin, Con A, and the biotin‐binding protein avidin‐peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case.     

The protonated Schiff base of halorhodopsin from Natronobacterium pharaonis is hydrolyzed at elevated temperatures

Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70 degrees C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl-concentration and reveals an anion binding site (in addition to the Cl- in the SB vicinity) with a binding constant of 100mM (measured at 70 degrees C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl-pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl- anion by Br-, I- and SCN- exhibits similar behavior to that of CI- in the region of 100mM but higher concentrations are needed for N3-, HCOO- and NO2-to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25 degrees C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.     

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.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

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.     

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells

Structure determination of biomacromolecules under in‐cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site‐directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well‐suited for in‐cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in‐cell trityl–iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal‐to‐noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in‐cell measurement.     

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.     

Protein-protein interaction of a Pharaonis halorhodopsin mutant forming a complex with Pharaonis halobacterial transducer protein II detected by Fourier-transform infrared spectroscopy

Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199(ppR)-Asn74(pHtrII) and Thr189(ppR)-Glu43 (pHtrII), Ser62(pHtrII). It was reported that a pHR mutant (P240T/F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L(1) and L(2) intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250(pHR) and Asn74(pHtrII) were detected for the L(1) and L(2) intermediates, supporting that Tyr250(pHR) forms a hydrogen bond with Asn74(pHtrII) as similarly to Tyr199(ppR). The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156(pHR) in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle.     

Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label

The nitroxide-containing nucleoside ?m is reported as the first rigid spin label for paramagnetic modification of RNA by solid-phase synthesis. The spin label is well accommodated in several RNA secondary structures as judged by its minor effect on the thermodynamic stability of hairpin and duplex RNA. Electron paramagnetic resonance (EPR) spectroscopic characterization of mono-, bi-, and trimolecular RNA structures shows that ?m will be applicable for advanced EPR studies to elucidate structural and dynamic aspects of folded RNA.     

Interaction of meso-tetrakis (4-sulfonatophenyl) porphyrin with cationic CTAC micelles investigated by small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR)

Small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been used to investigate the interaction of the water-soluble meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS(4)) with cationic cethyltrimethylammonium chloride (CTAC) micelles. To evaluate if the porphyrin protonation state affects its interaction with the micelle, both SAXS and EPR measurements were performed at pH 4.0 and 9.0. The best-fit SAXS curves were obtained assuming for CTAC micelle a prolate ellipsoidal shape in the absence and upon incorporation of 2-10 mM TPPS(4). SAXS results show that the presence of porphyrin impacts on micellar hydrophobic core, leading to a micellar reassembling into smaller micelles. Lineshapes of EPR spectra of 5- and 16-doxyl stearic acids (5- and 16-DSA, respectively) bound to 100 mM CTAC micelles exhibited slight changes as a function of porphyrin concentration. Spectral simulations revealed an increase of mobility restriction for both spin probes, especially at higher porphyrin concentration, where a small reduction of environment polarity was also observed for 16-DSA. The spin labels monitored only slight differences between pH 4.0 and 9.0, in agreement with the SAXS results.     

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.     

Spectroscopic characterizations of bridging cysteine ligand variants of an engineered Cu2(Scys)2 CuA azurin

Bridging cysteine ligands of the Cu(A) center in an engineered Cu(A) azurin were replaced with serine, and the variants (Cys116Ser and Cys112Ser Cu(A) azurin) were characterized by mass spectrometry, as well as UV-vis and electron paramagnetic resonance (EPR) spectroscopic techniques. The replacements resulted in dramatically perturbed spectroscopic properties, indicating that the cysteines play a critical role in maintaining the structural integrity of the Cu center. The replacements at different cysteine residues resulted in different perturbations, even though the two cysteines are geometrically symmetrical in the primary coordination sphere with respect to the two copper ions. The Cys112Ser variant contains two distinct type 2 copper centers, while the Cys116Ser variant has one type 1 copper center with slight tetragonal distortion. Both the UV-vis and EPR spectra of the Cys116Ser variant change with pH, and the pK(a) of the transition is 6.0. A type 1 copper EPR spectrum with A(||) = 26 G was obtained at pH 7.0, while a type 2 copper EPR spectrum with A(||) = 140 G was found at pH 5.0. Interestingly, lowering the temperature from 290 to 85 K resulted in conversion of the Cys116Ser variant from a type 1 copper center to a type 2 copper center, suggesting rearrangement of the ligand around the copper or binding of an exogenous ligand at low temperature. This difference in mutation effects at different cysteines may be due to different constraints exerted on the two cysteines by hydrogen-bonding patterns in the ligand loop.     

Stearic acid spin labels in lipid bilayers: insight through atomistic simulations

Spin-labeled stearic acid species are commonly used for electron paramagnetic resonance (EPR) studies of cell membranes to investigate phase transitions, fluidity, and other physical properties. In this paper, we use large-scale molecular dynamics simulations to investigate the position and behavior of nitroxide spin labels attached to stearic acid molecules in dipalmitoylphosphatidylcholine (DPPC) bilayers. The results of these studies are potentially very important for the interpretation of EPR spectra, which rely on assumptions about the position of the label in the membrane. Additionally, we investigate the effect of chirality and ionization of the carboxyl group of the label. For a non-ionized species, we observe that spin-label molecules are even able to make flip-flop transitions between the leaflets of the bilayer. Such transitions have been previously observed only in very rare cases in molecular simulations.     

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near‐room temperature. This approach is demonstrated using fullerene–nitroxide and fullerene–triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide‐labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.     

Similar membrane affinity of mono- and Di-S-acylated ras membrane anchors: a new twist in the role of protein lipidation

The functionally required membrane attachment of Ras is achieved through an invariant isoprenylation of a C-terminal Cys, supplemented by further lipid modification of adjacent Cys residues by one (N-ras) or two (H-ras) palmitoyls. However, whether the triply lipidated membrane anchor of H-ras has a higher membrane affinity than its doubly lipidated counterpart, or whether the affinity contribution of the two palmitates and the farnesyl is additive, was not known. To address this issue, we carried out potential of mean force (PMF or free energy profile) calculations on a hexadecylated but nonpalmitoylated anchor (Cys186-HD), hexadecylated and monopalmitoylated anchors (Cys181-monopalmitate and Cys184-monopalmitate), and a nonlipid-modified anchor. We found that the overall insertion free energy follows the trend Cys181/Cys184-bipalmitate (wild type) approximately Cys181-monopalmitate > Cys184-monopalmitate > nonpalmitoylated anchor. Consistent with suggestions from recent cell biological experiments, the computed PMFs, coupled with structural analysis, demonstrate that membrane affinity of the Ras anchor depends on both the hydrophobicity of the palmitate and the prenyl groups and the spacing between them. The data further suggest that while Cys181-palmitate and Cys186-farnesyl together provide sufficient hydrophobic force for tight membrane binding, the palmitoyl at Cys184 is likely designed to serve another, presumably functional, role.     

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.     

gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis,EPR Characterization,Rotamer Libraries and Biocompatibility

The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.     

Multiplexed Analysis of Peptide Functionality Using Lanthanide-based Structural Shift Reagents

Functionally selective lanthanide-based ion mobility shift reagents are presented as a method to elucidate protein or peptide structural information as well as relative quantitation of protein expression profiles. Sequence information and site localization of primary amines (n-terminus and lysine), phosphorylation sites, and cysteine residues can be obtained in a data dependent manner using ion mobility-mass spectrometry (IM-MS). The high mass of the incorporated lanthanide ensures a significant shift of where the signal occurs in IM-MS conformation space. Peptide sequence information provided by the use of IM-MS shift reagents allows for both a more confident identification of peptides from complex mixtures and site localization following tandem MS experiments. Stable isotopes of the lanthanide series may also be used as relative quantitation labels since several lanthanides can be utilized in differential sample analyses.     

Self-assembly of pluronic block copolymers in aqueous dispersions of single-wall carbon nanotubes as observed by spin probe EPR.

The self-assembly of Pluronic block copolymers in dispersions of single-wall carbon nanotubes (SWNT) was investigated by spin probe electron paramagnetic resonance (EPR) spectroscopy. Nitroxide spin labeled block copolymers derived from Pluronic L62 and P123 were introduced in minute amounts into the dispersions. X-band EPR spectra of the SWNT dispersions and of native polymer solutions were measured as a function of temperature. All spectra, below and above the critical micelle temperature (CMT), were characteristic of the fast limit motional regime. The temperature dependence of the 14N isotropic hyperfine coupling, aiso, and the rotational correlation time, tauc, were determined. It was observed that, below the CMT, EPR does not distinguish between chains adsorbed on SWNT and free chains. Above CMT, substantial differences were observed: in the native solution, the Pluronics spin labels experience only one environment, Sm, assigned to spin labels in the corona of the Pluronic micelle, whereas in the SWNT dispersions, in addition to Sm, a second population of nonaggregated, individual chains, Si, is observed. The relative amounts of Sm and Si were found to depend on the relative concentrations of the Pluronic and SWNT. Furthermore, the aggregates formed in the SWNT dispersions do not show the typical increase in chain-end mobility as a function of temperature, observed in the post-CMT regime of the native Pluronic solutions. This suggests a larger dynamical coupling among aggregated chains in the presence of the SWNT as compared to the native micelles. The overall findings are consistent with the formation of a new type of aggregates, composed of a SWNT-polymer hybrid.