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Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels. [ABSTRACT FROM AUTHOR]

Isothermal titration calorimetry experiments can provide significantly more detailed information about molecular interactions when combined in global analysis. For example, global analysis can improve the precision of binding affinity and enthalpy, and of possible linkage parameters, even for simple bimolecular interactions, and greatly facilitate the study of multi-site and multi-component systems with competition or cooperativity. A pre-requisite for global analysis is the departure from the traditional binding model, including an ‘ n ’-value describing unphysical, non-integral numbers of sites. Instead, concentration correction factors can be introduced to account for either errors in the concentration determination or for the presence of inactive fractions of material. SEDPHAT is a computer program that embeds these ideas and provides a graphical user interface for the seamless combination of biophysical experiments to be globally modeled with a large number of different binding models. It offers statistical tools for the rigorous determination of parameter errors, correlations, as well as advanced statistical functions for global ITC (gITC) and global multi-method analysis (GMMA). SEDPHAT will also take full advantage of error bars of individual titration data points determined with the unbiased integration software NITPIC. The present communication reviews principles and strategies of global analysis for ITC and its extension to GMMA in SEDPHAT. We will also introduce a new graphical tool for aiding experimental design by surveying the concentration space and generating simulated data sets, which can be subsequently statistically examined for their information content. This procedure can replace the ‘ c ’-value as an experimental design parameter, which ceases to be helpful for multi-site systems and in the context of gITC. [ABSTRACT FROM AUTHOR]

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum. [ABSTRACT FROM AUTHOR]

Abstract: Accurate measurements of rotor temperature are critical for the interpretation of hydrodynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor using iButton temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2°C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets. [Copyright &y& Elsevier]