Determination Of Rotational Correlation Times Research Articles

Rotational and translational dynamics and their relation to hydrogen bond lifetimes in an ionic liquid by means of NMR relaxation time experiments and molecular dynamics simulation.

We report a concerted theoretical and experimental effort to determine the reorientational dynamics as well as hydrogen bond lifetimes for the doubly ionic hydrogen bond +OH⋯O- in the ionic liquid (2-hydroxyethyl)trimethylammonium bis(trifluoromethylsulfonyl)imide [Ch][NTf2] by using a combination of NMR relaxation time experiments, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Due to fast proton exchange, the determination of rotational correlation times is challenging. For molecular liquids, 17O-enhanced proton relaxation time experiments have been used to determine the rotational correlation times for the OH vectors in water or alcohols. As an alternative to those expensive isotopic substitution experiments, we employed a recently introduced approach which is providing access to the rotational dynamics from a single NMR deuteron quadrupolar relaxation time experiment. Here, the deuteron quadrupole coupling constants (DQCCs) are obtained from a relation between the DQCC and the δ 1H proton chemical shifts determined from a set of DFT calculated clusters in combination with experimentally determined proton chemical shifts. The NMR-obtained rotational correlation times were compared to those obtained from MD simulations and then related to viscosities for testing the applicability of popular hydrodynamic models. In addition, hydrogen bond lifetimes were derived, using hydrogen bond population correlation functions computed from MD simulations. Here, two different time domains were observed: The short-time contributions to the hydrogen lifetimes and the reorientational correlation times have roughly the same size and are located in the picosecond range, whereas the long-time contributions decay with relaxation times in the nanosecond regime and are related to rather slow diffusion processes. The computed average hydrogen bond lifetime is dominated by the long-time process, highlighting the importance and longevity of hydrogen-bonded ion pairs in these ionic liquids.

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: a time-resolved fluorescence anisotropy study.

Self-assembly of HIV-1 integrase (IN) in solution has been studied previously by time-resolved fluorescence, using tryptophan anisotropy decay. This approach provides information on the size of macromolecules via the determination of rotational correlation times (theta). We have shown that, at submicromolar concentration, IN is characterized by a long rotational correlation time (theta(20 degrees C) = 90-100 ns) corresponding to a high-order oligomeric form, likely a tetramer. In the present work, we investigated the self-assembly properties of the DNA-bound IN by using three independent fluorophores. Under enzymatic assay conditions (10(-7) M IN, 2 x 10(-8) M DNA), using either fluorescein-labeled or fluorescent guanosine analog-containing oligonucleotides that mimic a viral end long terminal repeat sequence, we found that the DNA-IN complex was characterized by shorter theta(20 degrees C) values of 15.5-19.5 and 23-27 ns, calculated from experiments performed at 25 degrees C and 37 degrees C, respectively. These results were confirmed by monitoring the Trp anisotropy decay as a function of the DNA substrate concentration: the theta of IN shifted from 90-100 ns to lower values (<30 ns) upon increasing the DNA concentration. Again, the normalized theta(20 degrees C) values were significantly higher when monitored at 37 degrees C as compared with 25 degrees C. These results indicate that upon binding the viral DNA end, the multimeric enzyme undergoes a dissociation, most likely into a homogeneous monomeric form at 25 degrees C and into a monomer-dimer equilibrium at 37 degrees C.

Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation.

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin-spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of -170 +/- 15 ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1 ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6 ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived tau m values for proteins. The near identity of observed T2 and T1 rho values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine tau m values. It is also noted that the reliable determination of tau m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as tau m increases.

Luminescence studies of polyelectrolyte behaviour in solution: 2. The application of steady-state fluorescence anisotropy measurements to the study of the conformational behaviour of poly(methacrylic acid) in dilute aqueous solution

Steady-state anisotropy and fluorescence-quenching measurements have been used to study the segmental relaxation of poly(methacrylic acid) (PMAA) in dilute aqueous solution. None of the quenchers (CH 3NO 2, Tl + and I −) employed in this work exhibited truly dynamic quenching of the excited states of the fluorescent species (0.5 mol% copolymerized acenaphthylene and I-vinylnaphthalene) used to label the PMAA. As a consequence, the dependence of the measured anisotropy r upon the relative, quenched fluorescence intensity could not be used as a means of determination of the intrinsic anisotropy r 0. Furthermore, the influence of a static component in the quenching process rendered plots of r −1 as a function of fluorescence lifetime less reliable for estimation of r 0 than would be the case with a purely dynamic quencher. (This limitation is a direct consequence of the difficulties encountered in the measurement of r itself in the presence of significant static quenching.) Consequently, under these conditions, steady-state fluorescence anisotropy measurements have been shown to be of limited value in studying the conformational behaviour of PMAA. In contrast, time-resolved measurements have allowed the determination of rotational correlation times which are internally consistent and independent of the choice of fluorescent label at the pH values studied.

The potential of perturbed angular correlation of gamma rays as a tool for dynamic studies of peptides/proteins

Four peptides and serum albumin have been derivatized with the bicyclic anydride of diethylenetriaminepentaacetic acid. Procedures were developed to isolate the labelled species and determine the degree of derivatization. By using perturbed angular correlation of gamma-ray spectroscopy it is possible, through the determination of rotational correlation times, to decide whether labelled peptides interact with other molecules (receptors). In the case of the peptide cholecystokinin it is shown that the interaction between the peptide and its corresponding polyclonal antibody can be determined down to 1 pmol hormone. Experiments on 111In- and 111mCd-labelled Gly-Trp showed that, where the 111Cd PAC spectrum directly reflected the rotational motion of the molecule, the 111In PAC spectrum was affected by the nuclear transitions to 111mCd.

A simple method for determination of rotational correlation times and separation of rotational and polarity effects from EPR spectra of spin-labeled biomolecules in a wide correlation time range

A method using nitroxide radical spin labels for determining both the isotropic rotational correlation time τ R and the environmental polarity of the label is described. By means of a least square fitting method, the values of an effective hyperfine tensor A′ and of an effective g value tensor g′ of randomly oriented spin labels are determined from X-band EPR spectra on the basis of an effective time-independent Hamiltonian. The traces of the tensors deliver the information about the environmental polarity of the label and are not dependent on the rotational correlation time τ R. A new averaging parameter S ( τ R), calculated on the basis of the principal values of the tensor A′ , permits the evaluation of the rotational correlation time τ R in a very wide time range between 10 −10 and 10 −6 s. An application of this method to spin-labeled methemoglobin over a large temperature range and in environments of different polarity is discussed.

Determination of rotational correlation times from X- and Q-band spin-label EPR spectra of cytochrome P-450. Practical implications

A new method recently developed by J. S. Hyde and K. V. S. Rao ( J. Magn. Reson. 38, 313 (1980)) for the determination of rotational correlation times τ R in the slow-motional region is based on measuring EPR spin-label spectra at two different microwave frequencies. The utility of this method was proved by studying the dynamical state of spin-labeled substrate molecules bound to the endoplasmic hepatic cytochrome P-450 by use of X- and Q-band EPR spectroscopy. Under experimental conditions where only one slow-motional spin-label spectrum is observed, the new method exhibits advantages in comparison with other approaches. The experimental error in the determination of τ R however, increases significantly if (i) a free label spectrum is superimposed or (ii) the spin concentration (in many cases the protein concentration) is too low to produce a relatively noiseless Q-band spectrum. Membrane-bound proteins, e.g., maleimide spin-labeled liver microsomes, provide an appropriate reference system for such an analysis. The Hyde-Rao method is compared with a ST-EPR analysis of the same system.

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators.

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).

Construction and performance of a variable-frequency phase-modulation fluorometer.

We describe the construction and performance of a variable-frequency phase-modulation fluorometer. This instrument, which provides modulation frequencies from 1 to 200 MHz, was constructed using commercially available components. To facilitate the introduction of these instruments into other laboratories we describe in detail the chosen components and the principles of operation. The present light source is a continuous-wave helium-cadmium laser, which provides convenient excitation wavelengths of 325 and 442 nm. Modulation of the incident light is provided by one of several electro-optic modulators. The extent of modulation ranges from 1.0 to 0.2 as the frequency increases from 1 to 200 MHz. Phase angles and demodulation factors are measured using the cross-correlation method. The closely spaced frequencies are provided by two direct frequency synthesizers. The phase and modulation measurements are accurate to 0.2 degrees and 0.002, respectively, from 1 to 200 MHz. This accuracy allows considerable resolution of complex decay laws. The usefulness of frequency-domain fluorometry for the resolution of multiexponential decays is illustrated by the analysis of several difficult mixtures. As examples, we resolved a two-component mixture of anthracene (4.1 ns) and 9,10-diphenylanthracene (6.3 ns), and confirmed that the intensity decay of NADH in aqueous buffer is at least a double exponential (0.2 and 0.86 ns). We also resolved an especially difficult mixture of anthracene (4.1 ns) and 9-methylanthracene (4.5 ns), and a three-component mixture with decay times of 1.3, 4.1 and 7.7 ns. Frequency-domain fluorometers appear to be particularly useful for determination of complex decays of fluorescence anisotropy. This capability is illustrated by the determination of rotational correlation times as short as 47 ps for p-bis[2-(5-phenyloxazolyl)]benzene (POPOP) in hexane at 40 degrees C, and by the resolution of the two correlation times of anisotropic rotators such as perylene and 9-aminoacridine. Resolution of two anisotropy decay times for 9-aminoacridine is a difficult test because these correlation times differ by less than 2-fold. The resolution of multiexponential decays of intensity and anisotropy possible with this instrument is at least equivalent to that obtained using state-of-the-art time-resolved instruments based on mode-locked laser sources. The ease and rapidity of frequency-domain measurements, the relative simplicity of the equipment, the accuracy of the measurements and the lack of significant systematic errors indicate that frequency-domain fluorometry will be widely useful in chemical and biochemical research.

Studies of the molecular motions of large penetrant molecules in solid polymers by electron paramagnetic resonance spin-probe techniques

The determination of rotational correlation times for spin probes in polymers is reviewed, with emphasis on the relationships between rotational and translational motions. Data on the behavior of three different spin-probe molecules in polyamide and polyester films and fibers are examined in detail. It is concluded that saturation phenomena make it difficult to obtain reliable rigid limit parameters in these systems, unless very low microwave power levels can be used. At very low temperatures probe rotation appears to take place in local defects or microvoids in the glassy polymers. There is an increase in activation energy of rotation at a temperature close to the T g expected from dynamic mechanical (110 Hz) measurements, but the probe molecule rotations remain strongly hindered until significantly higher temperatures are reached. In nylons, sensitivity of probe rotations to structural differences between fibers is low in dry systems, and increases with temperature and moisture content. Even in nylon fibers containing substantial amounts of water, the probe rotation is most consistent with free-volume concepts, and is inconsistent with models of probe rotation and diffusion in solvent-filled pores or channels.

Direct determination of rotational correlation time by electron-spin echoes

The measurement of rotational correlation time by determination of the outer ERS hyperfine extrema is demonstrated for tempone in a glycerol-water solution.(AIP)

Determination of rotational correlation times from perturbed angular correlations of γ-rays. 111Indium bound to single-stranded DNA and DNA[Cu 2+

A rotational correlation time T c=62 +12 −10 ns was obtained for 111 In bound to single-stranded DNA at pH = 2.8. When Cu 2+ ions are bound to DNA, t c increases to 82 +5 −4 ns, indicating a higher rigidity for the DNA molecule.

Determination of rotational correlation time from perturbed angular correlation of .gamma. rays: apomyoglobin reconstituted with 111indium(III) mesoprotoporphyrin IX

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetermination of rotational correlation time from perturbed angular correlation of .gamma. rays: apomyoglobin reconstituted with 111indium(III) mesoprotoporphyrin IXAlan G. Marshall, K. M. Lee, and Peter W. MartinCite this: J. Am. Chem. Soc. 1980, 102, 4, 1460–1462Publication Date (Print):February 1, 1980Publication History Published online1 May 2002Published inissue 1 February 1980https://doi.org/10.1021/ja00524a060RIGHTS & PERMISSIONSArticle Views69Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (404 KB) Get e-Alerts Get e-Alerts

Determination of rotational correlation times of proteins in solution from carbon-13 spin-lattice relaxation measurements. Effect of magnetic field strength and anisotropic rotation.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetermination of rotational correlation times of proteins in solution from carbon-13 spin-lattice relaxation measurements. Effect of magnetic field strength and anisotropic rotationDavid J. Wilbur, Raymond S. Norton, Arthur O. Clouse, Robert Addleman, and Adam AllerhandCite this: J. Am. Chem. Soc. 1976, 98, 25, 8250–8254Publication Date (Print):December 1, 1976Publication History Published online1 May 2002Published inissue 1 December 1976https://doi.org/10.1021/ja00441a059RIGHTS & PERMISSIONSArticle Views206Altmetric-Citations43LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (529 KB) Get e-Alerts Get e-Alerts

A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 2: Determination of rotational correlation times of proteins in solution

Experiments have been performed to test the validity of measurements of slow rotational diffusion using eosin isothiocyanate as a probe. When eosin-protein conjugates are covalently bound to Sepharose 4B, the dichroism of the flash-induced absorption transient does not decay with time. The magnitude of the dichroism is smaller than for eosin in solid solution. These results, together with measurements with highly viscous solutions, demonstrate the existence of a rapid but restricted independent motion of the eosin probe. This causes a partial loss of pf dochroism leaving a residual dichroism whose time dependence is determined by the rotational motion of the protein. Rotational correlation times in the microsecond-millisecond time range have been determined for eosinprotein conjugates dissolved in glycerol-water mixtures of varying viscosity. The measured values are in good agreement with theoretical predictions and show the correct dependence on the viscosity of the medium and the size and state of association of the protein.

The determination of rotational correlation times in liquids from Raman bandshapes

A procedure is discussed and tested for extracting the rotational contribution to vibrational Raman bands in liquids. Molecular reorientation contributes only to the depolarized scattering so that the spectral distribution of the polarized part is used as a basis for removing all other effects on the bandshape. The method can be used successfully on polarized Raman bands and has been tested using glass-forming liquids for which the reorientation contribution disappears as the temperature is decreased to the glass transition. Reorientation times for benzene are compared with independent measurements.

Determination of rotational correlation time of nitric oxyde radicals in a viscous medium

The rotational correlation time of nitric oxyde radicals in a viscous medium have been determined with the use of the results of the theoretical ESR spectra calculations.

Factors involved in the determination of rotational correlation times for spin labels

The determination of rotational correlation times and activation energies by means of ESR line widths has been investigated in detail for di- t-butyl nitroxide and for four spin labels in solution. Consistent correlation times can be obtained only if the parameters b and Δγ in the spin Hamiltonian are determined separately for each radical solvent system. Line width corrections to account for unresolved hyperfine structure were found to affect significantly the apparent activation energies for rotation.