Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Clustering with hypergraphs: the case for large hyperedges

The extension of conventional clustering to hypergraph clustering, which involves higher order similarities instead of pairwise similarities, is increasingly gaining attention in computer vision. This is due to the fact that many grouping problems require an affinity measure that must involve a subset of data of size more than two, i.e., a hyperedge. Almost all previous works, however, have considered the smallest possible hyperedge size, due to a lack of study into the potential benefits of large hyperedges and effective algorithms to generate them. In this paper, we show that large hyperedges are better from both theoretical and empirical standpoints. We then propose a novel guided sampling strategy for large hyperedges, based on the concept of random cluster models. Our method can generate pure large hyperedges that significantly improve grouping accuracy without exponential increases in sampling costs. In the important applications of face clustering and motion segmentation, our method demonstrates substantially better accuracy and efficiency.Pulak Purkait, Tat-Jun Chin, Hanno Ackermann, David Sute

Salting-in with a salting-out agent : explaining the cation specific effects on the aqueous solubility of amino acids

Although the understanding of ion specific effects on the aqueous solubilities of biomolecules is crucial for the development of many areas of biochemistry and life sciences, a consensual and well-supported molecular picture of the phenomena has not yet been established. Mostly, the influence of cations and the nature of the molecular interactions responsible for the reversal of the Hofmeister trend in aqueous solutions of amino acids and proteins are still defectively understood. Aiming at contributing to the understanding of the molecular-level mechanisms governing the cation specific effects on the aqueous solubilities of biocompounds, experimental solubility measurements and classical molecular dynamics simulations were performed for aqueous solutions of three amino acids (alanine, valine, and isoleucine), in the presence of a series of inorganic salts. The evidence gathered suggests that the mechanism by which salting-in inducing cations operate in aqueous solutions of amino acids is different from that of anions, and allows for a novel and consistent molecular description of the effect of the cation on the solubility based on specific interactions of the cations with the negatively charged moieties of the biomolecules

Toward a Practical Method for Adaptive QM/MM Simulations

We present an accurate adaptive multiscale molecular dynamics method that will enable the detailed study of large molecular systems that mimic experiment. The method treats the reactive regions at the quantum mechanical level and the inactive environment regions at lower levels of accuracy, while at the same time molecules are allowed to flow across the border between active and environment regions. Among many other things, this scheme affords accurate investigation of chemical reactions in solution. A scheme like this ideally fulfills the key criteria applicable to all molecular dynamics simulations: energy conservation and computational efficiency. Approaches that fulfill both criteria can, however, result in complicated potential energy surfaces, creating rapid energy changes when the border between regions is crossed. With the difference-based adaptive solvation potential, a simple approach is introduced that meets the above requirements and reduces fast fluctuations in the potential to a minimum. In cases where none of the current adaptive QM/MM potentials are able to properly describe the system under investigation, we use a continuous force scheme instead, which, while no longer energy conserving, still retains a related conserved quantity along the trajectory. We show that this scheme does not introduce a significant temperature drift on time scales feasible for QM/MM simulations