Monitoring protein unfolding transitions by NMR-spectroscopy

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

Low-field 1H NMR relaxometry of water molecules confined in microporous UiO-66

Water molecules confined in a microporous metal–organic framework (MOF) UiO-66 are characterized by a low-field 1H nuclear magnetic resonance (NMR) spectroscopy. Measurements are performed of the longitudinal ([Formula: see text] and transverse ([Formula: see text] relaxation times as a function of water content from fully saturated to incomplete coverage of the first-adsorbed monolayer. The results obtained indicate that the relaxation of water molecules confined in UiO-66 is within the fast-exchange regime. When the amount of water exceeds filling ratio [Formula: see text] = 0.4, the averaged relaxation time is approximately linearly dependent on water filling ratio in pore. When the water amount cannot support a full coverage of surface monolayer, the relaxation rate increases with less filling ratio, illustrating that the mobility of water molecules is more restricted. Analysis of the measured values and the simulated ones leads to the conclusion that the surface-affected zone in UiO-66 is not confined to the surface monolayer.

Nongaussian Intravoxel Incoherent Motion Diffusion Weighted and Fast Exchange Regime Dynamic Contrast-Enhanced-MRI of Nasopharyngeal Carcinoma: Preliminary Study for Predicting Locoregional Failure

The aim of the present study was to identify whether the quantitative metrics from pre-treatment (TX) non-Gaussian intravoxel incoherent motion (NGIVIM) diffusion weighted (DW-) and fast exchange regime (FXR) dynamic contrast enhanced (DCE)-MRI can predict patients with locoregional failure (LRF) in nasopharyngeal carcinoma (NPC). Twenty-nine NPC patients underwent pre-TX DW- and DCE-MRI on a 3T MR scanner. DW imaging data from primary tumors were fitted to monoexponential (ADC) and NGIVIM (D, D*, f, and K) models. The metrics Ktrans, ve, and τi were estimated using the FXR model. Cumulative incidence (CI) analysis and Fine-Gray (FG) modeling were performed considering death as a competing risk. Mean ve values were significantly different between patients with and without LRF (p = 0.03). Mean f values showed a trend towards the difference between the groups (p = 0.08). Histograms exhibited inter primary tumor heterogeneity. The CI curves showed significant differences for the dichotomized cutoff value of ADC ≤ 0.68 × 10−3 (mm2/s), D ≤ 0.74 × 10−3 (mm2/s), and f ≤ 0.18 (p < 0.05). τi ≤ 0.89 (s) cutoff value showed borderline significance (p = 0.098). FG’s modeling showed a significant difference for the K cutoff value of ≤0.86 (p = 0.034). Results suggest that the role of pre-TX NGIVIM DW- and FXR DCE-MRI-derived metrics for predicting LRF in NPC than alone.

CALX-CBD1 Ca2+-binding cooperativity studied by NMR spectroscopy and ITC with Bayesian statistics

ABSTRACTThe Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologues, CALX is inhibited by Ca2+-binding to CALX-CBD1, while CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a β-sandwich and displays four Ca2+ binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+-binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift time scale. Ca2+-binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N CEST and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates (≈ 20 s−1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal due to the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill = 2.9 − 3.1, near maximum cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is at least three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+-binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+-concentration in sensory neuronal cells.SIGNIFICANCECALX-CBD1 is the Ca2+-sensor domain of the Na+/Ca2+ exchanger of Drosophila melanogaster. It consists of a β-sandwich, and contains four Ca2+ binding sites at the distal loops. In this study, we examined the cooperative binding of four Ca2+ ions to CALX-CBD1 using NMR spectroscopy and isothermal titration calorimetry (ITC) experiments. NMR and ITC data were analyzed using the framework of the binding polynomial formalism and Bayesian statistics. A novel approach to analyze NMR titration data in the slow exchange regime was proposed. These results support the view that CALX-CBD1 binds four Ca2+ with high cooperativity. The significant ligand binding cooperativity exhibited by this domain is determinant for the efficient allosteric regulation of this exchanger by intracellular Ca2+.

Spin Wave Modes in a Cylindrical Nanowire in Crossover of Dipolar-Exchange Regime

Although the magnetic wires have been [1] broadly investigated, some of their dynamicalproperties, like: (anti)crossing between the spin wave modes and the impact of the magnetic field onthe spin wave spectrum, still need to be explored. [...]

China’s Dollar-linked Hong Kong during the Global Crisis

This paper explores the domestic price level and trade competitiveness of Hong Kong in addition to the compatibility of this dollar-based currency board to the criteria inspired by the optimum currency area (OCA) theory. On price and competitiveness, findings point out that during the turmoil Hong Kong had not performed as well as that in the past and an apparent reason for this is the inflows of hot capital from abroad especially of the US that fuelled rising property prices. On conformity to the OCA criteria, the findings broadly corroborate the fixed exchange regime with the US as the monetary anchor country but at the same time China appears as a prospective contender to US as the monetary anchor. In the longer run, amidst the prolonged economic and monetary weaknesses in the US plus the emergence of renminbi as a global currency, Hong Kong might as well unify its exchange rate with the Chinese money.

Transient dynamics in strongly nonlinear systems: optimization of initial conditions on the resonant manifold

We consider a system of two linear and linearly coupled oscillators with ideal impact constraints. Primary resonant energy exchange is investigated by analysis of the slow flow using the action–angle (AA) formalism. Exact inversion of the action-energy dependence for the linear oscillator with impact constraints is not possible. This difficulty, typical for many models of nonlinear oscillators, is circumvented by matching the asymptotic expansions for the linear and impact limits. The obtained energy–action relation enables the complete analysis of the slow flow and the accurate description of the critical delocalization transition. The transition from the localization regime to the energy-exchange regime is captured by prediction of the critical coupling value. Accurate prediction of the delocalization transition requires a detailed account of the coupling energy with appropriate redefinition and optimization of the limiting phase trajectory on the resonant manifold. This article is part of the theme issue ‘Nonlinear energy transfer in dynamical and acoustical systems’.