Cytoskeletal protein kinases: titin and its relations in mechanosensing

Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation

Effect of acute hypoxia on maximal oxygen uptake and maximal performance during leg and upper-body exercise in Nordic combined skiers

We examined the effect of normobaric hypoxia (3200 m) on maximal oxygen uptake (VO2max) and maximal power output (Pmax) during leg and upper-body exercise to identify functional and structural correlates of the variability in the decrement of VO2max (DeltaVO2max) and of maximal power output (DeltaPmax). Seven well trained male Nordic combined skiers performed incremental exercise tests to exhaustion on a cycle ergometer (leg exercise) and on a custom built doublepoling ergometer for cross-country skiing (upper-body exercise). Tests were carried out in normoxia (560 m) and normobaric hypoxia (3200 m); biopsies were taken from m. deltoideus. DeltaVO2max was not significantly different between leg (-9.1+/-4.9%) and upper-body exercise (-7.9+/-5.8%). By contrast, Pmax was significantly more reduced during leg exercise (-17.3+/-3.3%) than during upper-body exercise (-9.6+/-6.4%, p<0.05). Correlation analysis did not reveal any significant relationship between leg and upper-body exercise neither for DeltaVO2max nor for DeltaPmax. Furthermore, no relationship was observed between individual DeltaVO2max and DeltaPmax. Analysis of structural data of m. deltoideus revealed a significant correlation between capillary density and DeltaPmax (R=-0.80, p=0.03), as well as between volume density of mitochondria and DeltaPmax (R=-0.75, p=0.05). In conclusion, it seems that VO2max and Pmax are differently affected by hypoxia. The ability to tolerate hypoxia is a characteristic of the individual depending in part on the exercise mode. We present evidence that athletes with a high capillarity and a high muscular oxidative capacity are more sensitive to hypoxia

Cross-polarization for dissolution dynamic nuclear polarization

Dynamic nuclear polarization (DNP) in combination with subsequent dissolution of the sample allows the detection of low-γ nuclei in the solution state with a signal gain of up to tens of thousand times compared to experiments starting from Boltzmann conditions. The long polarization build-up times of typically more than one hour are a drawback of this technique. The combination of dissolution DNP with cross-polarization (CP) in the solid state was shown to have the potential to overcome this disadvantage. In this article we discuss the cross-polarization step under dissolution DNP conditions in more detail. We show that adiabatic half-passage pulses allow us to enhance the CP efficiency in power-limited DNP probes. As a low-power alternative to Hartmann–Hahn CP we also demonstrate the applicability of frequency-swept de- and re-magnetization pulses for polarization transfer via dipolar order. We investigate the implications and restrictions of the common solid-state DNP mechanisms to the DNP–CP technique and apply a spin-thermodynamic model based on the thermal-mixing mechanism. The model allows us to investigate the dynamics of the polarization levels in a system with two nuclear Zeeman reservoirs and explains the enhanced DNP efficiency upon solvent deuteration within a spin-thermodynamic picture

Dissolution dynamic nuclear polarization efficiency enhanced by Hartmann–Hahn cross polarization

Dynamic nuclear polarization (DNP) can be used to increase the polarization of nuclei by utilizing the higher electron polarization. Typically, in dissolution DNP heteronuclei are directly polarized due to their longer relaxation times. We present the combination of DNP to protons with subsequent cross polarization to 13C followed by dissolution to solution state. The experiment was implemented using a modified dissolution DNP probe. For a [13C]urea sample doped with TEMPO the carbon polarization and its build-up rate could be increased by a factor of two using CP. With small losses, this gain could be transferred to the solution by dissolution

Dissolution DNP using trityl radicals at 7 T field

Characterization of direct 13C DNP at 1.4 K and 7 T field using trityl radicals

Highly stable magic angle spinning spherical rotors

The use of spherical rotors for magic angle spinning offers a number of advantages, including improved sample exchange, efficient microwave coupling for dynamic nuclear polarization nuclear magnetic resonance (NMR) experiments, and, most significantly, high frequency and stable spinning with minimal risk of rotor crash. Here we demonstrate the simple retrofitting of a commercial NMR probe with MAS spheres for solid-state NMR. We analyze a series of turbine groove geometries to investigate the importance of the rotor surface for spinning performance. Of note, rotors lacking any surface modification spin rapidly and stably even without feedback control. The high stability of a spherical rotor about the magic angle is shown to be dependent on its inertia tensor rather than the presence of turbine grooves.</p

Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

ISSN:0925-2738ISSN:1573-500