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PURPOSE: This work aims to unravel the intricacies of adiabatic rotating frame relaxometry in biological tissues. THEORY AND METHODS: The classical formalisms of dipolar relaxation R 1 ρ $$ {R}_{1\rho } $$ and R 2 ρ $$ {R}_{2\rho } $$ were systematically analyzed for water molecules reorienting on "fast" and "slow" timescales. These two timescales are, respectively, responsible for the absence and presence of R 1 ρ $$ {R}_{1\rho } $$ dispersion. A time-averaged R 1 ρ $$ {R}_{1\rho } $$ or R 2 ρ $$ {R}_{2\rho } $$ over an adiabatic pulse duration was recast into a sum of R 1 $$ {R}_1 $$ and R 2 $$ {R}_2 $$ , but with different weightings. These weightings depend on the specific modulations of adiabatic pulse waveforms. In this context, stretched hyperbolic secant ( HSn $$ HSn $$ ) pulses were characterized. Previously published H S 1 $$ HS1 $$ R 1 ρ $$ {R}_{1\rho } $$ , continuous-wave (CW) R 1 ρ $$ {R}_{1\rho } $$ , and R 1 $$ {R}_1 $$ measures from 12 agarose phantoms were used to validate the theoretical predictions. A similar validation was also performed on previously published HSn $$ HSn $$ R 1 ρ $$ {R}_{1\rho } $$ ( n $$ n $$ =1, 4, 8) and HS 1 $$ HS1 $$ R 2 ρ $$ {R}_{2\rho } $$ from bovine cartilage specimens. RESULTS: Longitudinal relaxation weighting decreases for HSn $$ HSn $$ pulses as n $$ n $$ increases. Predicted CW R 1 ρ cal $$ {R}_{1\rho}^{cal} $$ values from agarose phantoms align well with the measured CW R 1 ρ exp $$ {R}_{1\rho}^{exp} $$ values, as indicated by a linear regression function: R 1 ρ cal = 1.04 * R 1 ρ exp - 1.96 $$ {R}_{1\rho}^{cal}={1.04}^{\ast }{R}_{1\rho}^{exp}-1.96 $$ . The predicted adiabatic R 1 ρ $$ {R}_{1\rho } $$ and R 2 ρ $$ {R}_{2\rho } $$ from cartilage specimens are consistent with those previously measured, as quantified by: R 1 ρ , 2 ρ cal = 1.10 * R 1 ρ , 2 ρ exp - 0.41 $$ {R}_{1\rho, 2\rho}^{cal}={1.10}^{\ast }{R}_{1\rho, 2\rho}^{exp}-0.41 $$ . CONCLUSION: This work has theoretically and experimentally demonstrated that adiabatic R 1 ρ $$ {R}_{1\rho } $$ and R 2 ρ $$ {R}_{2\rho } $$ can be recast into a sum of R 1 $$ {R}_1 $$ and R 2 $$ {R}_2 $$ , with varying weightings. Therefore, any suggestions that adiabatic rotating frame relaxometry in biological tissues could provide more information than the standard R 1 $$ {R}_1 $$ and R 2 $$ {R}_2 $$ warrant closer scrutiny.

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Electrospun ultrathin fibers based on binary compositions of polylactide (PLA) and poly(ε-caprolactone) (PCL) with the various content from the polymer ratio from 0/100 to 100/0 have been explored. Combining thermal (DSC) and spectropy (ESR) techniques, the effect of biopolymer content on the characteristics of the crystal structure of PLA and PCL and the rotative diffusion of the stable TEMPO radical in the intercrystallite areas of PLA/PCL compositions was shown. It was revealed that after PLA and PCL blending, significant changes in the degree of crystallinity of PLA, PCL segment mobility, sorption of the Tempo probe, as well as its activation energy of rotation in the intercrystalline areas of PLA/PCL fibers, were evaluated. The characteristic region of biopolymers' composition from 50/50 to 30/70% PLA/PCL blend ratio was found, where the inversion transition of PLA from dispersive medium to dispersive phase where an inversion transition is assumed when the continuous medium of the PLA transforms into a discrete phase. The performed studies made it possible, firstly, to carry out a detailed study of the effect of the system component ratio on the structural and dynamic characteristics of the PLA/PCL film material at the molecular level.

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Intrinsically disordered proteins (IDPs) defy the conventional structure-function paradigm by lacking a well-defined tertiary structure and exhibiting inherent flexibility. This flexibility leads to distinctive spin relaxation modes, reflecting isolated and specific motions within individual peptide planes. In this work, we propose a new pulse sequence to measure the longitudinal 13C' CSA-13C'-13Cα DD CCR rate [Formula: see text] and present a novel 3D version of the transverse [Formula: see text] CCR rate, adopting the symmetrical reconversion approach. We combined these rates with the analogous ΓxyN/NH and ΓzN/NH CCR rates to derive residue-specific correlation times for both spin-pairs within the same peptide plane. The presented approach offers a straightforward and intuitive way to compare the correlation times of two different and complementary spin vectors, anticipated to be a valuable aid to determine IDPs backbone dihedral angles distributions. We performed the proposed experiments on two systems: a folded protein ubiquitin and Coturnix japonica osteopontin, a prototypical IDP. Comparative analyses of the results show that the correlation times of different residues vary more for IDPs than globular proteins, indicating that the dynamics of IDPs is largely heterogeneous and dominated by local fluctuations.

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The hydration of ions and charge groups controls electro mass transfer through ion exchange systems. The self-diffusion and local mobility of water molecules as well as lithium cations in poly (4-styrenesulfonic acid) and its lithium, sodium and cesium salts were investigated for the first time using pulsed-field gradient NMR (PFG NMR) and NMR relaxation techniques. The temperature dependences of the water molecule and Li+ cation self-diffusion coefficients exhibited increasing self-diffusion activation energy in temperature regions below 0 °C, which is not due to the freezing of parts of the water. The self-diffusion coefficients of water molecules and lithium cations, as measured using PFG NMR, are in good agreement with the self-diffusion coefficients calculated based on Einstein's equation using correlation times obtained from spin-lattice relaxation data. It was shown that macroscopic water molecules' and lithium cations' transfer is controlled by local particles jumping between neighboring sulfonated groups. These results are similar to the behavior of water and cations in sulfonic cation exchanger membranes and resins. It was concluded that polystyrenesulfonic acid is appropriate model of the ionogenic part of membranes based on this polymer.

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The interconnection of ionogenic channel structure, cation hydration, water and ionic translational mobility was revealed in Nafion and MSC membranes based on polyethylene and grafted sulfonated polystyrene. A local mobility of Li+, Na+ and Cs+ cations and water molecules was estimated via the 1H, 7Li, 23Na and 133Cs spin relaxation technique. The calculated cation and water molecule self-diffusion coefficients were compared with experimental values measured using pulsed field gradient NMR. It was shown that macroscopic mass transfer is controlled by molecule and ion motion near sulfonate groups. Lithium and sodium cations whose hydrated energy is higher than water hydrogen bond energy move together with water molecules. Cesium cations in possession of low hydrated energy are directly jumping between neighboring sulfonate groups. Cation Li+, Na+ and Cs+ hydration numbers (h) in membranes were calculated from 1H chemical shift water molecule temperature dependences. The values calculated from the Nernst-Einstein equation and the experimental conductivity values were close to each other in Nafion membranes. In MSC membranes, calculated conductivities were one order of magnitude more compared to the experimental ones, which is explained by the heterogeneity of the membrane pore and channel system.

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Significance: Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore's diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, θ imaging is still rarely investigated in general and in carbon dots (CDs) in particular. Aim: To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and θ, together with the steady-state images of fluorescence intensity (FI) and FA (r). Approach: The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. Results: The FLT of fluorescein samples was found to decrease from 4.01±0.01 to 3.56±0.02 ns, whereas both r and θ were significantly increased from 0.053±0.012 to 0.252±0.003 and 0.15±0.05 to 11.25±1.87 ns, respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of r from 0.100±0.011 to 0.150±0.013 and θ from 0.98±0.13 to 1.65±0.20 ns for the first CDs and from 0.280±0.008 to 0.310±0.004 and 5.55±1.08 to 7.95±0.97 ns for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs. Conclusions: Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, r, and θ). Nevertheless, θ was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

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Cholesterol is the most representative sterol present in membranes of higher eukaryotes, and is the end product of a long and multistep biosynthetic pathway. Lathosterol and zymosterol are biosynthetic precursors of cholesterol in Kandutsch-Russell and Bloch pathways, respectively. Lathosterol differs with cholesterol merely in the position of the double bond in the sterol ring, whereas zymosterol differs with cholesterol in position and number of double bonds. In this work, we have monitored the effect of cholesterol and its biosynthetic precursors (lathosterol and zymosterol) on membrane organization and dynamics in fluid and gel phase membranes. Toward this goal, we have utilized two fluorescent membrane probes, DPH and its cationic derivative TMA-DPH. Our results using these probes show that cholesterol and its biosynthetic precursors (lathosterol and zymosterol) exhibit similar trend in maintaining membrane organization and dynamics (as reported by fluorescence anisotropy and apparent rotational correlation time), in fluid phase POPC membranes. Notably, although lathosterol and zymosterol show similar trend in maintaining membrane organization and dynamics, the corresponding change for cholesterol is different in gel phase DPPC membranes. These results demonstrate that the position and number of double bonds in sterols is an important determinant in maintaining membrane physical properties. Our results assume significance since accumulation of precursors of cholesterol have been reported to be associated with severe pathological conditions.

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Degeneration of cartilage can be studied non-invasively with quantitative MRI. A promising parameter for detecting early osteoarthritis in articular cartilage is T1ρ, which can be tuned via the amplitude of the spin-lock pulse. By measuring T1ρ at several spin-lock amplitudes, the dispersion of T1ρ is obtained. The aim of this study is to find out if the dispersion contains diagnostically relevant information complementary to a T1ρ measurement at a single spin-lock amplitude. To this end, five differently acquired dispersion parameters are utilized; A, B, τc, T1ρ/T2, and R2 - R1ρ. An open dataset of an equine model of post-traumatic cartilage was utilized to assess the T1ρ dispersion parameters for the evaluation of cartilage degeneration. Firstly, the parameters were compared for their sensitivity in detecting degenerative changes. Secondly, the relationship of the dispersion parameters to histological and biomechanical reference parameters was studied. Parameters A, T1ρ/T2, and R2 - R1ρ were found to be sensitive to lesion-induced changes in the cartilage within sample. Strong correlations of several dispersion parameters with optical density, as well as with collagen fibril angle were found. Most of the dispersion parameters correlated strongly with individual T1ρ values. The results suggest that dispersion parameters can in some cases provide a more accurate description of the biochemical composition of cartilage as compared to conventional MRI parameters. However, in most cases the information given by the dispersion parameters is more of a refinement than complementary to conventional quantitative MRI.

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Since the first membrane models in the 1970s, the concept of biological membranes has evolved considerably. The membrane is now seen as a very complex mixture whose dynamic behavior is even more complex. Solid-state NMR is well suited for such studies as it can probe the movements of the membrane from picoseconds to seconds. Two NMR observables can be used: motionally averaged spectra and relaxation times. They bring information on order parameters, phase transitions, correlation times, activation energies and membrane elasticity. Spectra are used to determine the nature of the membrane phase. The order parameters can be measured directly from spectra that are dominated by quadrupolar, dipolar and chemical shielding magnetic interactions and allow describing the lipid membrane as being very rigid at the glycerol and chain level and very fluid at its center and surface. Correlation times and activation energies can be measured for intramolecular motions (pico to nanoseconds), molecular motions (nano to 100 ns) and collective modes of membrane deformation (microseconds). Sterols modulate membrane phases, order parameters, correlation times and membrane elasticity. In general terms, sterols tend to act to reduce the impact of environmental changes on molecular order and dynamics. They can be described as regulators of membrane dynamics by keeping them in a state of dynamics that changes very little when the temperature or other factors change. The presence of such large-scale membrane dynamics is proposed as a means of adapting to evolutionary constraints.

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Choline-based deep eutectic solvents (DESs) are potential candidates to replace flammable organic solvent electrolytes in lithium-ion batteries (LIBs). The effect of the addition of a lithium salt on the structure and dynamics of the material needs to be clarified before it enters the battery. Here, the archetypical DES choline chloride:urea at 1:2 mole fraction has been added with lithium chloride at two different concentrations and the effect of the additional cation has been evaluated with respect to the non-doped system via multinuclear NMR techniques. 1H and 7Li spin-lattice relaxation times and diffusion coefficients have been measured between 298 K and 373 K and revealed a decrease in both rotational and translational mobility of the species after LiCl doping at a given temperature. Temperature dependent 35Cl linewidths reflect the viscosity increase upon LiCl addition, yet keep track of the lithium complexation. Quantitative indicators such as correlation times and activation energies give indirect insights into the intermolecular interactions of the mixtures, while lithium single-jump distance and transference number shed light into the lithium transport, being then of help in the design of future DES electrolytes.

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Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores.

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Starch paste is a very complex dispersion that cannot be clearly classified as a solution, colloid or suspension and many factors affects its properties. As these ambiguities constitute a barrier to technological development, the aim of this study was to investigate the interaction of starch macromolecules with water by analysing the results of rheological properties, low field nuclear magnetic resonance (LF NMR), dynamic light scattering (DLS) and ζ potential analyses. Starch pastes with a concentration of 1%, prepared with distilled water and buffered to pH values of 2.5, 7.0 and 9.5 were analysed. It was proved that the pH buffering substantially decreased the values of consistency index but the pH value itself was not significant. LF NMR studies indicated that the dissolution of starch in water resulted in a reduction in spin-lattice as well as spin-spin relaxation times. Moreover, changes in relaxation times followed the patterns observed in rheological studies. Electrokinetic and DLS analyses showed that potential values are primarily influenced by the properties of the starches themselves and, to a lesser extent, by the environmental conditions. The conducted research also showed complementarity and, to some extent, substitutability of the applied research methods as well as exclusion chromatography (a method not used in this work).

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Nuclear magnetic resonance (NMR) techniques are widely used to identify pure substances and probe protein dynamics. Edible oil is a complex mixture composed of hydrocarbons, which have a wide range of molecular size distribution. In this research, low-field NMR (LF-NMR) relaxation characteristic data from various sample oils were analyzed. We also suggest a new method for predicting the size of edible oil molecules using LF-NMR relaxation time. According to the relative molecular mass, the carbon chain length and the transverse relaxation time of different sample oils, combined with oil viscosity and other factors, the relationship between carbon chain length and transverse relaxation time rate was analyzed. Various oils and fats in the mixed fluid were displayed, reflecting the composition information of different oils. We further studied the correlation between the rotation correlation time and the molecular information of oil molecules. The molecular composition of the resulting fluid determines its properties, such as viscosity and phase behavior. The results show that low-field NMR can obtain information on the composition, macromolecular aggregation and molecular dynamics of complex fluids. The measurements of grease in the free-fluid state show that the relaxation time can reflect the intrinsic properties of the fluid. It is shown that the composition characteristics and states of complex fluids can be measured using low-field nuclear magnetic resonance.

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The structure and dynamics of cefpodoxime proxetil are elucidated by measuring chemical shift anisotropy (CSA) tensor, spin-lattice relaxation time, and local correlation time at twenty-one crystallographically different 13C nuclei sites. The principal components of CSA tensor of cefpodoxime proxetil are extracted by the two-dimensional phase adjusted sinning sideband (2DPASS) cross-polarization magic angle spinning (CP-MAS) solid-state NMR experiment, and the spin-lattice relaxation time is measured by the method outlined by Torchia(T1CP). The local correlation time is calculated by bearing in mind that the spin-lattice relaxation mechanism of 13C nuclei is mainly governed by the CSA interaction and the heteronuclear dipole-dipole interaction. The aminothiazole ring, ß-lactam ring, and dihydrothiazine ring provide stability to the drug molecule and increase the affinity of the drug to penicillin-binding proteins (PBPs) receptors. The principal components of CSA parameters, spin-lattice relaxation time, and local correlation time vary substantially for carbon nuclei residing on these three rings. These signify that not only the electronic environment, but the molecular conformation, and the local dynamics are also altered within the ring. The substitution of the acyl side chain, oxime group, and the aminothiazole ring at the C7 position of the ß-lactam ring enhances the antibacterial activity and the binding affinity of the drug. A huge variation of the spin-lattice relaxation time and local correlation time is observed in those regions. The change in the electron charge distribution and nuclear spin dynamics at different parts of the drug molecule is clear by CSA and spin-lattice relaxation measurements, which will enrich the field "NMR crystallography".

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The biocompatible, biodegradable, linear copolymer sodium alginate is fabricated from [Formula: see text] linked [Formula: see text]-D-mannuronic acid (M block) and [Formula: see text]-L-guluronic acid (G-block). It has wide applications in drug delivery, cell encapsulation, and commercial application in the textile, cosmetics, paper, food, biomedical, and pharmaceutical industries. The structure and dynamics of sodium alginate were here investigated by measuring chemical shift anisotropy (CSA) parameters, spin-lattice relaxation time, and molecular correlation time. The principal components of the CSA tensor were determined by two-dimensional phase-adjusted spinning sideband (2DPASS) cross-polarization magic angle spinning (CP-MAS) SSNMR. The alternating M and G blocks of both equatorial and axial links are associated with greater overall flexibility. The molecular correlation time of the carboxyl carbon of both G and M blocks is faster than for the anomeric carbon and pyranose carbon. This is further experimental evidence of the coexistence of two different dynamics within the polysaccharide chains of sodium alginate, which was previously established by 1H-13C dipolar profile analysis. The relaxation time of the para-crystalline region of sodium alginate is comparable with that of chitosan, but it is much shorter than that of cellulose and chitin. The order of the molecular correlation time of sodium alginate and chitosan is also the same. Hence, it can be concluded that sodium alginate exhibits greater flexibility than cellulose and chitin. These types of investigation into the local electronic configuration and nuclear spin dynamics at various carbon nuclei sites of the biopolymer at atomic-scale resolution will help in the design of biomimetic materials.

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PURPOSE: MR R2 imaging of ordered tissue exhibits the magic angle effect, potentially masking subtle pathological changes in cartilage. This work aimed to develop an orientation-independent order parameter (S) exclusively sensitive to collagen degeneration. METHODS: A theory was developed based on R1ρ dispersion coupled with a simplified molecular motion model in which anisotropic R2a(θ) became directly proportional to correlation time τbθ and S could be derived. This new parameter was validated with ex vivo R1ρ dispersion reported on orientated (n = 4), enzymatically depleted bovine cartilage (n = 6), and osteoarthritic human knee specimens (n = 14) at 9.4 Tesla, which was further demonstrated on 1 healthy human knee in vivo at 3 Tesla. RESULTS: τbθ from orientation-dependent R1ρ dispersion revealed a significantly high average correlation (r = 0.89 ± 0.05, P < 0.05) with R2a (θ) on cartilage samples and a moderate correlation (r = 0.48, P < 0.001) for the human knee in vivo. The derived S (10-3 ) significantly decreased in advanced osteoarthritis (1.64 ± 0.03 vs. 2.30 ± 0.11, P < 0.001) and collagen-depleted samples (1.30 ± 0.11 vs. 2.12 ± 0.12, P < 0.001) when compared with early osteoarthritis and the control, respectively. CONCLUSION: The proposed order parameter could be a potentially useful orientation-independent MR biomarker for collagen alterations in cartilage and other highly structured tissues.

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Internal structure and dynamics of commercial and natural cellulose were studied by measuring chemical shift anisotropy (CSA) parameters, and spin-lattice relaxation rate (1/T1) at each and every chemically different carbon nuclear site. CSA parameters were measured by 13C two-dimensional phase adjusted spinning sideband (2DPASS) cross-polarization magic angle spinning (CP-MAS) NMR experiment. Site specific spin-lattice relaxation time was measured by Torchia-CP method. Anisotropy parameters of C4 and C6 regions are higher than C1 and C235 regions and asymmetry of C4 line is lower than any other carbon site. The higher values of CSA parameters of C4 and C6 nuclei arise due to the rotation of O4-C4, C1-O4, O5-C5-C6-O6 and C4-C5-C6-O6 bonds at torsion angles ψ, Φ, χ and χ' respectively and the influence of interchain and intrachain hydrogen bondings. Two distinct peaks are also observed for C4 and C6 resonance line position-one peak arises primarily due to the nuclei in amorphous region and another one arises due to the same nuclei resides in paracrystalline region. The spin-lattice relaxation time and the CSA parameters are different at these two distinct peak positions of C4 and C6 line. Molecular correlation time of each and every chemically different carbon site was calculated with the help of CSA parameters and spin-lattice relaxation time. The molecular correlation time of the amorphous region is one order of magnitude less than the crystalline region. The distinction between amorphous and paracrystalline regions of cellulose is more vividly portrayed by determining spin-lattice relaxation time, CSA parameters, and molecular correlation time at each and every chemically different carbon site. This type of study correlating the structure and dynamics of cellulose will illuminate the path of inventing biomimetic materials.

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Structure and dynamics of natural and regenerated chicken feather ß-keratin were investigated by 13C cross-polarization (CP) magic angle spinning (MAS) solid state nuclear magnetic resonance (SSNMR) spectral analysis, 13C and 1H spin-lattice relaxation time measurements, and 13C two dimensional phase adjusted spinning sidebands (2DPASS) MAS SSNMR measurements. Chemical shift anisotropy (CSA) parameters of both natural and regenerated chicken feather ß-keratin were extracted by using 2DPASS MAS SSNMR experiment. The beauty of 2DPASS MAS SSNMR experiment is it can correlate the isotropic and anisotropic dimension with the help of shearing transformation and two dimensional Fourier Transformation. Molecular correlation time at each and every magnetically inequivalent carbon site of both natural and regenerated chicken feather ß-keratin were also determined. The change in molecular dynamics of structural protein after pretreatment was monitored by 2DPASS MAS SSNMR and 13C relaxation measurement. This type of comprehensive study will provide the information about the interrelation between the structure and dynamics of structural protein and will also shed light in the way of developing methods for conversion of animal by-products to novel product.

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Nuclear magnetic resonance (NMR) relaxation data and molecular dynamics (MD) simulations are combined to characterize the dynamics of the fungal prion HET-s(218-289) in its amyloid form. NMR data is analyzed with the dynamics detector method, which yields timescale-specific information. An analogous analysis is performed on MD trajectories. Because specific MD predictions can be verified as agreeing with the NMR data, MD was used for further interpretation of NMR results: for the different timescales, cross-correlation coefficients were derived to quantify the correlation of the motion between different residues. Short timescales are the result of very local motions, while longer timescales are found for longer-range correlated motion. Similar trends on ns- and µs-timescales suggest that µs motion in fibrils is the result of motion correlated over many fibril layers.

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Fluorescence depolarization kinetics measured by the time-resolved fluorescence anisotropy decay serves as a sensitive and powerful methodology to study the conformational dynamics of macromolecules. This methodology allows us to delineate the different modes of biomolecular motional dynamics including the local, segmental, and global rotational dynamics on the timescale ranging from picoseconds to nanoseconds. In this chapter, we describe the principles and applications of this methodology to obtain unique molecular insights into the intrinsically disordered proteins (IDPs). Fluorescence depolarization kinetics, when performed in a site-specific manner, can offer a reliable tool to monitor the intrinsic backbone torsional dynamics of expanded IDPs and is capable of discerning the conformational preference of IDPs. Additionally, the time-resolved fluorescence anisotropy measurements allow us to investigate the mechanism of binding and assembly of a wide range of IDPs that are involved in crucial function and disease.

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