The tuning of the pKa of ionizable residues plays a critical role in various protein functions, such as ligand-binding, catalysis, and allostery. Proteins harness the free energy of folding to position ionizable groups in highly specific environments that strongly affect their pKa values. To investigate the interplay among protein folding kinetics, thermodynamics, and pKa modulation, we introduced a pair of Asp residues at neighboring interior positions of a coiled-coil. A single Asp residue was replaced for an Asn side chain at the a-position of the coiled-coil from GCN4, which was also crosslinked at the C-terminus via a flexible disulfide bond. The thermodynamic and kinetic stability of the system was measured by circular dichroism and stopped-flow fluorescence as a function of pH and concentration of guanidine HCl. Both sets of data are consistent with a two-state equilibrium between fully folded and unfolded forms. Distinct pKa values of 6.3 and 5.35 are assigned to the first and second protonation of the Asp pair; together they represent an energetic difference of 5 kcal/mol relative to the protonation of two Asp residues with unperturbed pKa values. Analysis of the rate data as a function of pH and denaturant concentration allowed calculation of the kinetic constants for the conformational transitions of the peptide with the Asp residues in the doubly protonated, singly protonated, and unprotonated forms. The doubly and singly protonated forms fold rapidly, and a ϕ-value analysis shows that their contribution to folding occurs subsequent to the transition state ensemble for folding. By contrast, the doubly charged state shows a reduced rate of folding and a ϕ-value near 0.5 indicative of a repulsive interaction, and possibly also heterogeneity in the transition state ensemble. 相似文献
Abstract: A systematic account of highly diverse cyrtocrinid faunules from Upper Jurassic strata of ?tramberk type (Oxfordian–Tithonian) in southern Poland (Polish Carpathians) is presented. Fourteen taxa (Phyllocrinus malbosianus, Ph. stellaris, Ph. sp., Psalidocrinus armatus, Sclerocrinus compressus, S. polonicus sp. nov., Hemicrinus aff. kabanovi, Ancepsicrinus parvus gen. et sp. nov., Tetracrinus baumilleri sp. nov., Eugeniacrinites alexandrowiczi, E. cf. moravicus, E. sp., Eudesicrinus gluchowskii sp. nov. and Hemibrachiocrinus tithonicus sp. nov. are described and illustrated. Representatives of the genus Eudesicrinus, previously recorded only from the Lower Jurassic, are here shown to extend into the uppermost Jurassic. Other cyrtocrinids considered are common in Jurassic/Cretaceous strata across Europe. In the present faunules, isocrinid (Isocrinida), comatulid (Comatulida) and roveacrinid (Roveacrinida sensu Rasmussen, inclusive of Saccocoma) crinoids are associated. 相似文献
A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity. 相似文献
Two stable and water-soluble organometallic carbonyl cluster derivatives have been prepared and shown to enter the cell with ease. The CO stretching vibrations afford strong mid-infrared signals which have been demonstrated, for the first time, to be of utility in cell imaging via an IR microscope. 相似文献
Tea is the most common beverage after water. Concerns have been raised about the safety of tea during pregnancy, especially for embryo development. We aimed at studying the effects of active tea components on developing embryos by in vitro rat embryo culture. Rat embryos during early organogenesis were cultivated in serum supplemented with one of the tea catechins. Developmental hallmarks and malformations (Mal) in the developing embryos were compared and evaluated by a standard morphological scoring system. The embryotoxicity of each tea catechin was classified according to the European Center for the Validation of Alternative Methods. Cell viability was assessed by supervital dye staining, apoptosis by TUNEL assay, and peroxidation by the 8-isoprostane EIA method. We found that (+)-catechin had the least effect on developing embryos (Mal(50)=715.1 mg/L; IC50(Mal)=435 mg/L), whereas (-)-epigallocatechin gallate had the most adverse effect (Mal(50)=54.2 mg/L; IC50(Mal)=45.8 mg/L). The major malformation in affected embryos included caudal retardation with abnormal axial flexion and delayed hind-limb formation. All catechins were classified as nonembryotoxic except (-)-epigallocatechin gallate, which was classified as weakly embryotoxic. With (-)-epigallocatechin gallate, increased numbers of nonviable and apoptotic cells in the malformed embryos were associated with increased embryo 8-isoprostane. 相似文献
Heating, ventilation and air conditioning (HVAC) systems are widely used to regulate indoor temperature and air quality of modern buildings. The central supply and exhaust system cause the dispersal and removal of bioaerosols. This study presents results from ex situ experiments conducted to a better understanding of the spatiotemporal distribution, passive dispersal and removal of fungal spores through HVAC systems. The study was conducted in a 50 square meters by 3 m high instrument room in a research building with HVAC systems. Plates with Aspergillus flavus colony were attached upside down on the edge of the ceiling vent to imitate the mildew growing. Fungal spores were released for 10 min, collected and counted by the settle plate method in 2 h. Results show that A. flavus spores dispersed to 3.6 m in 2 min and were evenly distributed in the room within 8 min. The concentration of spores decreased from 48 to 3–6 CFU/plate after 60 min and was lower than 1% after 120 min. Our results suggest that airborne fungal spores disperse and remove by HVAC systems efficiently. Without the external and internal source, the HVAC system could greatly reduce the fungal amount in the indoor air to the background level within one hour. This study provided the observed data of the transmission and retention of internal or external biological contaminants through HVAC system.