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The helix-loop-helix (i.e., EF-hand) is the most common motif in the superfamily of Ca2+-binding proteins. Parvalbumins, as the classical EF-hand proteins, are associated with the muscle relaxation/contration, the calcium buffering etc. The previous researches focus more of interest on the ion coupled/decoupled mechanism using molecular dynamics (MD) computations. We developed the novel approach instead of MD, in which the Landau free energy was introduced to describe the protein in term of the skeletal Cα chain. The unfolding and folding processes were simulated by the Glauber algorithm under the repeated heating and cooling cycles. The high-quality crystal structure (2PVB) was as the trigger of non-equilibrium dynamics simulation for parvalbumin-β (Parv). The evolution of three dimensional structure during the folding was displayed for the residues 8-64 fragment in Parv. The phenomenon of helix nucleation was observed, that was, the 310 helix at residues 35-37 and the front of α-helix at residues 40-50 were firstly formed in the folding process. We also found two potential misfoldings which were caused by the distortion of the local structures at residues 35-37, 45-50. [ABSTRACT FROM AUTHOR]

Mus musculus centrin 1 ( MmCen1) is located at the cilium of photoreceptor cells connecting the outer segment through signal transduction components to the metabolically active inner segment. In the cilium, MmCen1 is involved in the translocation of transducin between compartments as a result of photoreceptor activation. In this study, we report the crystal structure of wild-type MmCen1 and its Ca-binding properties using structure-based mutagenesis. The crystal structure exhibits three structural features, i.e. four Ca equally occupied at each EF-hand motif, structural changes accompanying helix motion at the N- and C-lobes, and adoption of N-C type dimerization when Ca binds to EF-hand I and II in the N-lobe. The presence of MmCen1 dimers was confirmed in solution by native PAGE. Isothermal titration calorimetry data showed sequential binding of Ca at four independent sites. Mutations S45A and D49A in EF-hand I alone disrupted the Ca-binding property of the wild-type protein. Based on the crystal structure of MmCen1, we suggest that a dimerization mode between the N- and C-lobes may be required by Ca binding at the N-lobe. [ABSTRACT FROM AUTHOR]

Human phospholipid scramblase 1 (hPLSCR1) scrambles plasma membrane phospholipids during cell activation, blood coagulation and apoptosis. It was over-expressed in E. coli with a histidine tag and purified from the inclusion bodies (~30 mg/l culture broth) under denaturing conditions using 8 M urea. The denatured hPLSCR1 refolded into its native configuration when urea was removed as shown by a 10-fold increase in its intrinsic fluorescence. Active hPLSCR1 showed scrambling activity in vitro after reconstituting in proteoliposomes. hPLSCR1 showed higher rates of scrambling activity for phosphatidylethanolamine than phosphatidylcholine. Binding studies with the calcium analogue “Stains-all” dye showed a characteristic peak, termed as the J band, at 650 nm. This is the first report on high level expression of hPLSCR1 with histidine tag in E. coli. [ABSTRACT FROM AUTHOR]

TSD2, a gene necessary for DNA synthesis in Ustilago maydis, was cloned by complementation of the temperature sensitive growth defect of a mutant known previously as pol1-1 and renamed here tsd2-1. Linkage analysis established that the cloned fragment contained an allele of tsd2-1 and not a suppressor. DNA sequence determination of the cloned DNA fragment indicated the presence of a single large uninterrupted open reading frame capable of encoding a protein of 845 amino acids without homology to any known gene involved in DNA synthesis. TSD2 was found to be cell cycle-regulated and mRNA levels peaked in early S or G1 phase. [ABSTRACT FROM AUTHOR]