Evaluation of Conformational dynamics properties of spin-labelled proteins at different degrees of nitroxide radicals immobilization

Spin-labelling has found wide applications in elucidation of the dynamic behaviour of biological macromolecules in aqueous media and biomembranes. Most of the proposed methods aimed at estimation of macromolecular correlation times (τ_c) assume, however, spin label molecules rigidly bound within the protein matrix. To avoid this limitation theoretical models which involve additional dynamic parameters to characterize the spin label motion should be considered. We have used ESR spectra analysis technique which permits quantitative separation of slow macromolecular rotation (described by the rotational correlation time, τ_c) and fast anisotropic relative to protein nitroxyl radical motion (described by the “order parameter”,S). This method was applied to study: i) conformational dynamics of covalently and non-covalently spin-labelled human serum albumin (HSA) in solution; ii) protein-protein (antigen-antibody) interactions in a model system containing spin-labelled bovine serum albumin (BSA) and anti-BSA immunoglobulin (IgG) in solution; and iii) dynamic properties of membrane-bound proteins: H⁺-ATPase (CF₁-CF₀ coupling factor of photophosphorylation) and Photosystem I pigment-protein reaction centre complex (PSI RC) isolated from spinach chloroplasts and reconstituted in proteoliposomes.     

Thermodynamically Induced Conformational Changes of the Cyanobacterial Circadian Clock Protein KaiB

Site-directed spin labeling electron spin resonance (ESR) was applied to investigate the local environment of the cyanobacterial circadian clock protein KaiB. We prepared five cysteine residue-substituted mutants of KaiB labeled with maleimide spin label (MSL). By comparing the ESR spectra of KaiBs carrying MSL at different positions (Thr64, Lys67, Tyr94, Gly98, and Ala101), local conformational changes were identified. The ESR spectra of MSL-T64C and MSL-K67C showed the relatively slow motion of MSL characterized by τ?=?79 and 59?ns at 4°C, respectively. The spectra of MSL-Y94C, MSL-G98C and MSL-A101C showed relatively fast motion characterized by τ?=?8.0, 4.1 and 3.1?ns at 4°C, respectively. These differences were explained by the local environments of the position in KaiB. On incubation at 40°C for 24?h, all ESR spectra of the labeled KaiBs changed, which can be explained by the structural relaxation of KaiB.     

An electron spin resonance study of the soy bean phosphatidylcholine-based reversed micelles

The ESR spectra of l-palmitoyl-2-stearoyl-(n-doxyl)-glycero-3-phosphocholine spin label positional isomers (n = 5, 7, 10, 12 and 16) have been studied in soy bean phosphatidylcholine (SPC)-based microemulsions with various volume fractions of disperse phase over the wide temperature range. The maximum hyperfine splitting 2A _max and the order parametersS were taken as indices of the rotational mobility and the motion spatial restrictions of the labeled lipid chain segments. It is found that the temperaturesT _tr at which sharp enhancements of2A _max andS occur depend on concentration and size of the reversed micelles in solutions. To explain this, a plausible model, taking into account capability of the SPC molecule hydrocarbon chains to change a tilt angle with respect to the surface of a polar head group as temperature varies, is proposed. The estimations of the correlation times τ_sl obtained from the lineshape characteristics of the ESR spectra provided the possibility to suggest that these correlation times characterize the reorientations of the SPC chain axis about the normal to the surface of a polar head group of a reversed micelle.     

Electron spin relaxation studies of La@C82 in the solid state by EPR

The temperature dependence of EPR spectrum of La@C₈₂ in the powder of empty C₆₀ and C₇₀ mixed crystals was studied by EPR spectroscopy employing X- and Q-band microwave frequencies. The rigid limit spectra (at 4.2 K for the X-band and at 132 K for the Q-band) could be analyzed by static spectral simulation which yielded the EPR parameters,g _‖=2.0021,g _⊥=1.9970,^La A _⊥=7.8 MHz,^La A _‖～0 MHz and an isotropic¹³C coupling value of about 3 MHz. For higher temperatures an appreciable motional averaging effect was observed and the spectra were analyzed by using dynamic spectral simulation based on the stochastic Liouville equation, where we assumed an isotropic rotational motion with the Brownian diffusion. The calculated spectra reproduced the dominant feature of the temperature dependence of the spectra almost satisfactorily for both the X-and Q-band frequencies with the appropriate rotational correlation times. The Arrhenius plots of the correlation time gave two activation energies of 0.9 kcal/mol and 2.9–3.8 kcal/mol for the temperatures below and above 200 K, respectively.     

Dynamics of liquid ammonia from cold neutron scattering

The scattering of 4.1 Å neutrons by liquid ammonia has been measured at 218 K in the angular range of 30–90° using the Trombay rotating crystal spectrometer. The experimental data, after correcting for multiple scattering, have been compared with model calculations, and it is shown that it is possible to get detailed information about the rotational correlation function on the basis of neutron experiments alone. The model assumes Langevin diffusion for translational motions. Rotational motions are described by means of an orientational correlation function which has a gaseous behaviour for times up toτ ₀ and then changes over to a diffusive character with a rotational diffusion constantD _r. Within the framework of the model the correlation function can be described withD _r=0.28×10¹³sec^?1 andτ ₀=0.57×10^?18sec. Corrections for multiple scattering and their dependence on model parameters are discussed.     

Reorientational motion of acetone in solutions

Dipolar correlation functions of acetone in various solvents are obtained by Fourier transform of infrared spectra. The analysis of the shapes of correlation functions shows that the reorientational process is diffusional in the time scale longer than about 0.3 × 10^?12 sec. In nonpolar solvents the ratios of correlation times τ_A : τ_B : τ_C do not vary in different solvents, and the reorientation around b axis is more restricted than around the other axis. The length of correlation time is dependent on the density of solvent rather than the viscosity. These observations are unexpected if only the electrostatic interaction is considered. In polar solvents correlation times are longer than in nonpolar solvents and the tendency is especially remarkable in τ_B. An electrostatic interaction between polar molecules seems to be responsible for these observations. The solvent effect on the frequency shifts of carbonyl stretching vibration is consistent with the result of correlation times.     

Effect ofV K centers on positron annihilation in KCl and Ag-doped KCl

Positron lifetime spectra have been measured at 77 K for KCl and Ag-doped KCl before and after x-irradiation at 77 K and after annealing at room temperature. Radiation at 77 K reduces the intensity of the intermediate lifetime (τ₂) component. Radiation-induced defects were monitored optically and by ESR. The experiment shows that the changes observed in the positron decay are associated with the presence ofV _K centers.     

Electric dipole moments and nuclear hyperfine interactions for H2S,HDS, and D2S

Electric dipole moments and nuclear hyperfine coupling constants for several low-energy rotational states of the ground vibrational state of H₂S, HDS, and D₂S have been determined by molecular beam electric resonance spectroscopy. Analysis of the Stark effects for radiofrequency spectra of the lowest rotational levels gives dipole moments of 0.978325 (10) D for H₂S (J = 1, τ = 0), 0.977294 (20) D for D₂S (J = 1, τ = 0), and 0.977484 (60) D for HDS (J = 1, τ = 1 and J = 2, τ = 0). The electric field gradient tensor at the deuterium nucleus, the nuclear spin-spin tensor, and the spin-rotation tensor were evaluated from the hyperfine components of the radiofrequency spectra. The accuracy of these tensors was moderately improved over earlier measurements. From the H₂S spin-rotation tensor, the average paramagnetic shielding for the protons was found to be σ_av^p = ?104.84 (40) ppm. Combining this result with the proton NMR absolute chemical shift allowed the average diamagnetic shielding to be calculated as σ_av^d = 135.7 (5) ppm.     

Microwave spectroscopic study of the conformations,relative energies,and dipole moments of ethyl cyanoformate

The microwave spectrum of ethyl cyanoformate displays a-type band spectra from three nearly prolate conformers. High-resolution spectra of the two more stable species have been assigned. One form, designated extended, has rotational constants A″ = 6453.3(4) MHz, B″ = 1500.47(6) MHz, C″ = 1236.36(6) MHz, which are consistent with a syn-anti [τ₁ (OCOC) = 0°, τ₂ (COCC) = 180°] structure. The second form, labeled compact, has rotational constants A″ = 6787.8(7) MHz, B″ = 1549.38(8) MHz, C″ = 1406.80(8) MHz, which are consistent with a syn-gauche [τ₁ (OCOC) = 0°, τ₂ (COCC) ～ 90°] structure. The extended form is marginally more stable, ΔE = 55 ± 27 cm^?1. The extended conformer has dipole moment components μ_a = 4.44(7), μ_b ～ 0 D and the compact conformer has dipole moment components μ_a = 4.25(7), μ_b = 0, μ_c = 1.08(23) D. The third conformer (relative energy 600 ± 140 cm^?1) has the most intense band series even at ?63°C. the bands of this conformer are unresolvable into individual rotational transitions.     

Lipid chain mobility in interdigitated DPPC systems

The segmental lipid chain mobility in the gel phase of dipalmitoylphosphatidylcholine (DPPC) multilayers dispersed in buffer and in the interdigitated gel phase induced by glycerol, ethylene glycol, ethanol and chaotropic salt NaClO₄ was compared by using conventional electron spin resonance (ESR) spectroscopy. The stearic acids bearing the nitroxide moiety at different positions down the acyl chain (n-NSA,n-5, 7, 10, 12 and 16) were used to characterized the chain motion, and the outer hyperfine splittings of the spectra, 2A _max, were taken as indices of the rotational mobility of the chain in the gel phase. The ESR measurements revealed a gradient of increased mobility on proceeding towards the terminal methyl end in the fully hydrated gel phase of DPPC bilayers. This gradient was reduced in the interdigitated gel phase induced by ethanol and chaotropic salt NaClO₄, whereas the rotational mobility throughout the length of the chain was comparable to that near the polar/apolar interface in the interdigitated gel phase in glycerol and ethylene glycol. Moreover, the motional anisotropy was much less affected by temperature in the interdigitated gel state of DPPC in glycerol and ethylene glycol as compared both to normal bilayer gel phase and to the other interdigitated DPPC systems. Finally, there was no evidence for chain interdigitation in the fluid phase of DPPC dispersions in any medium.     

ESR-Linienbreite der Ionen der Eisengruppe in Lösungen

Electron spin resonance linewidths of ions belonging to the first transition group with quenched orbital angular momentum are calculated using a modified relaxationmatrix theory including third and fourth order perturbation terms. We base our calculation on a Hamiltonian, which depends on electron spin, nuclear spin, orbital angular momentum, rotation of the whole complex, and vibration of the ligands. Quadrupol effects, intermolecular electron-electron and electron-nucleus interactions are neglected. The results show that the well-known formula of the transverse relaxation time derived by using the spin-Hamiltonian is correct, if first the contribution of the rotational spin orbit process is taken in consideration and second the rotational correlation timeτ _c is replaced byτ _v =(1/τ _c +1/τ(0))^?1. 1/τ(0) describes the linewidth of the lowest energy value of the electrostatic energy of unpaired electrons in the ligand field. The linewidth arises from the normal modes of the complex; the calculation gives τ(0)=l0^?11...10^?12 sec.     

Lifetime measurement in the α system (C3Δ3-X3Δ3) of TiO

The radiative lifetimes of two rotational levels of the v′ = 2 level of the C³Δ₃ state of TiO have been measured using laser-induced fluorescence and delayed coincidence counting. Individual rotational levels are excited and observed. The measured lifetimes are τ_{J = 17} = 28.21 + 0.15 nsec and τ_{J = 87} = 29.74 ± 0.86 nsec. Argon-TiO collision cross sections have been determined for the two rotational levels and are found to differ by 30%.     

Analysis of fluorescence quenching of pyronin B and pyronin Y by molecular oxygen in aqueous solution

The fluorescence quenching of pyronin B and pyronin Y molecules by molecular oxygen in aqueous solution was studied by using steady-state and time-resolved fluorescence and UV-Vis absorption spectroscopy techniques. In order to understand the quenching mechanism, fluorescence decays, absorption and fluorescence spectra of the probes were recorded as a function of the oxygen concentration and temperature. The quenching was found to be appreciable and shows positive deviation in the Stern-Volmer representation obtained from the fluorescence intensity ratio. Fluorescence quenching constants (k_q) were calculated from the τ_o/τ vs. [Q] plots having linear correlation and compared with calculated diffusion-controlled rate constants (k_diff) values. Experimental results were in good agreement with the simultaneous dynamic and static quenching model.     

Upper and lower bounds of quartic centrifugal distortion constants

Expressions are obtained for the maximum and minimum possible values of the equilibrium centrifugal distortion constants τ_αβγδ for a molecule with given structure and vibrational frequencies. The method of calculation, based on Lagrangian multipliers, is also applied to the empirical centrifugal constants (such as D_J, D_JK, D_K, or Δ_J, Δ_JK, Δ_K, δ_J, δ_K) employed in fitting the rotational structure of spectra. These formulas make it possible to test whether a set of observed centrifugal constants is consistent with the rotational constants and vibrational frequencies. When inconsistencies arise, they are probably due to zero-point vibrational effects. By means of a second method employing inequalities, simpler formulas are derived for wider bounds. These are useful in calculating approximate values of the constants, as a preliminary to deciding which terms to include in the rotational Hamiltonian of any particular molecule.     

Low temperature protein dynamics studied by spin-labelling methods

Spin-labelling method in combination with modern versions of ESR spectroscopy allows one to study intramolecular dynamics of protein globules and surrounding molecules of water in a wide range of correlation times (τ _c = 10²?10^?11 s) and amplitudes and phonon processes as well. Experimental results for a number of proteins at temperatures from 30 to 300 K and at various humidity are in favour of glass-like dynamical model of proteins.     

Permeability Studies of Redox-Sensitive Nitroxyl Spin Probes Through Lipid Membranes Using an L-Band ESR Spectrometer

Electron spin resonance (ESR) studies were carried out for 2 mM ¹⁴N-labeled deuterated 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) and 3-carboxy-2,2,5,5,-tetramethyl-pyrrolidin-1-oxyl (carboxy-PROXYL) in pure water and various concentrations of liposomal solution by using an L-band ESR spectrometer. The ESR parameters, such as the line width, hyperfine coupling constant, g-factor, rotational correlation time and partition parameter, were reported for the samples. The changes in the line width were observed for ¹⁴N-labeled deuterated MC-PROXYL and carboxy-PROXYL in liposomal solution. The hyperfine coupling constant was observed for both nitroxyl spin probes. The permeable and impermeable nature of nitroxyl radicals was demonstrated using the ESR L-band spectra. The rotational correlation time increases with increasing concentration of liposome. The partition parameter for ¹⁴N-labeled deuterated MC-PROXYL in liposomal solution increases with increasing concentration of liposome, which reveals that the nitroxyl spin probe permeates into lipid membrane. The lipid peaks were observed for 2 mM ¹⁴N-labeled deuterated MC-PROXYL in 200, 300 and 400 mM liposomal concentration. The lipid peaks were not observed for ¹⁴N-labeled deuterated carboxy-PROXYL. These results indicate the permeable and impermeable nature of ¹⁴N-labeled deuterated nitroxyl spin probe.     

ESR of O-3 trapped in γ-irradiated NaClO3

The ESR spectra of γ-irradiated NaClO₃ was analysed at Q-Band, between 110 to 300 K. The single crystals were annealed at 180° C until the spectra indicated the presence of only one paramagnetic center. This center was indentified as the ozonide O^-₃ with $\text{g}\text{}\text{= 2.007±0.001}$, $\text{g}\text{⊥}\text{= 2.010±0.001}$ and g_av=2.009. Four magnetically inequivalent sites per unit cell with symmetry axes along the cube diagonals were observed. The axial symmetry found for the g-tensor was interpreted in terms of molecule rotation around the trigonal axes. A rotational frequency of 2×10⁹ c/s and an activation energy of 10 mev for the rotational motion could be estimated.     

Electron transfer at lanthanum endohedral fullerenes

The aim of this contribution is to study lanthanum containing fullerenes produced and treated under different conditions. The work was done using solid soot extract giving poorly resolved ESR spectra or separated lanthanofullerenes in solution showing well resolved ESR spectra. ESR experiments in connection with the mass spectrometry give information on the electronic states due to the electron transfer from the endohedral metal to the fullerene cage and the influence of oxygen on endohedral lanthanofullerene. The analysis of ESR spectra indicates the presence of up to nine octets of lanthanum containing fullerenes. These species have different stabilities. Furthermore, a very narrow single ESR line in the lanthanofullerene spectrum was detected having a linewidth ΔB=0.008 mT and ag-factor 2.0025. The electron transfer to the endohedral La@C₈₂ molecule studied by voltammetry and electrochemicalin situ ESR experiments is described. The interaction of the lanthanofullerene with oxygen in solution is also studied. In different solvents the influence of oxygen on line broadening in the ESR spectra of La@C₈₂ was observed.     

Pulsed field ionisation—ZEKE photoelectron spectrum of o-, m- and p-tolunitrile

Pulsed field ionisation—ZEKE photoelectron spectroscopy has been applied to o-, m- and p-tolunitrile in a supersonic jet. The PFI-ZEKE photoelectron spectra of m- and p-tolunitrile show well-resolved anharmonic structures in the low-frequency region, which are assigned to bands due to internal rotational motion of the methyl group in the cation. Level energies and relative transition intensities are reproduced well by a one-dimensional rotor model with a three-fold axis potential. Potential curves for the internal rotation have been determined. For o-tolunitrile, no band due to internal rotation was found in PFI-ZEKE spectrum. It is suggested that the o-tolunitrile cation has the high barrier for internal rotation and the stable conformation that is the same as that in S₁ and S₀. The barrier height and the conformation are compared with other toluene derivatives, and the relation between the electronic character of –CN and the internal rotational motion has been discussed.     

Solution Structure of Proline Pentapeptide. Analysis of the NOESY Spectra by Means of Spectrum Simulation

In this paper the NOESY spectra of proline pentapeptide are analyzed, and computer-simulated NOESY spectra are compared with the experimental spectra themselves. In simulating the spectra, the full relaxation matrix method is applied without assuming slow- or fast-motion limits. The calculated spectra are fitted to the experimental one in the following four steps: (1) determination of the proton-proton distance constraints from the experimental NOESY spectrum; (2) calculation of a rough 3D molecular structure by means of a distance geometry algorithm (DISGEO); (3) refinement of the structure; (4) adjustment of the correlation time of the overall tumbling motion of the molecule. The obtained proline pentapeptide structure in solution is close to the all-trans configuration of poly-L-prolines and the correlation time of the overall tumbling motion of the molecule is 0.4 ns; i.e. the isotropic rotational diffusion constant is D = 4.2 × 10⁸ s⁻¹.