Dependence of the kinetics of singlet-singlet energy transfer on spectral overlap

Dependence of the kinetics of singlet-singlet energy transfer on spectral overlap. the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a in the family (5,C7). Similar to other picornaviruses, EV71 contains a single-stranded, positive-sense RNA encoding a large polyprotein precursor (8, 9). The polyprotein is further cleaved into four structural proteins (VP1 to VP4) to form the viral capsid and seven nonstructural proteins (2A to 3D) for virus replication via the 2A protease and 3C protease (10, 11). Except for the cleavage of VP1/2A by the 2A protease (12) and the RNA-dependent cleavage of VP2/4 (13), the 3C protease is absolutely required for the cleavage of other junction sites within the polyprotein (14,C16). Meanwhile, EV71 3C reportedly interferes with the polyadenylation of host cell RNA by digesting CstF-64, a critical host factor for 3 pre-mRNA processing, suggesting a novel mechanism by which picornaviruses utilize 3Cpro to impair host cell function (17). In addition, the 3C protease can also cleave numerous factors and regulators associated with AKT1 cellular DNA-dependent RNA polymerases I, II, and III, such as the octamer-binding protein (OCT-1), TATA box-binding protein (TBP), cyclic AMP-responsive element-binding protein (CREB), transcription activator p53, histone H3, and DNA polymerase III (18,C21). The pivotal role of 3C protease in EV71 replication makes it an attractive target for antiviral discovery (22). The crystal structure of unliganded EV71 3C protease showed that EV71 3C protease folded into two domains that are related to other picornaviral 3C protease structures (23). The complex structures of EV71 mutants H133G, E71A, E71D with the inhibitor rupintrivir are similar to that of the unliganded protease structure (24). Lu et al. thoroughly characterized the 3C proteases from EV71 and CVA16 and reported a series of structures of both enzymes in free, peptide-bound, or inhibitor-bound form (25). These findings offered exact molecular insights into the substrate acknowledgement and inhibition of 3C protease. Profiling of the EV71 3C protease substrate could not only provide in-depth knowledge of catalytic mechanism at a molecular level, which would facilitate the design of potent protease inhibitors, but also lead to SL910102 development of a reliable and powerful biochemical assay for screening. In 2008, Kuo et al. synthesized six dodecapeptide substrates derived from the EV71 protease cleavage site and one dodecapeptide substrate (TSAVLQSGFRKM) from your severe acute respiratory syndrome coronavirus (SARS-CoV) protease autoprocessing site for biochemical characterization of the EV71 3C protease by determining their specificities using high-performance liquid chromatography (HPLC). The results showed that EV71 3C protease cleaved TSAVLQSGFRKM more efficiently than the additional six substrates (26). Eight peptides derived from CVA16 polyprotein and three peptides derived from EV71 polyprotein were also investigated for his or her susceptibilities to 3C cleavage via HPLC assay, and the peptide (IGNTIEALFQGPPKER) related to 2C-3A junction site of CVA16 could be efficiently processed by both proteases (= 8.37 M/min for EV71 and 10.72 M/min for CVA16) (25). However, the HPLC assay is limited to conveniently determine the substrate specification, and the technique of fluorescence resonance energy transfer (FRET) is commonly utilized for the preparation of fluorogenic substrates for biochemical characterization of proteases and protease inhibitor screening (27). A fluorogenic peptide, = 5.8 M; and of 7.1 10?4 M?1 min?1 (23), indicating that the peptide Dabcyl-RTATVQGPSLDFKE-Edans may not be the optimal substrate for EV71 protease inhibitor testing. The aim of this study was to biochemically characterize EV71 3C protease with an aim to develop a reliable and efficient assay for the screening of EV71 3C protease inhibitors. The.The nonspecific contaminants were then removed by washing with 20 column volumes of lysis buffer. additional picornaviruses, EV71 contains a single-stranded, positive-sense RNA encoding a large polyprotein precursor (8, 9). The polyprotein is definitely further cleaved into four structural proteins (VP1 to VP4) to form the viral capsid and seven nonstructural proteins (2A to 3D) for disease replication via the 2A protease and 3C protease (10, 11). Except for the cleavage of VP1/2A from the 2A protease (12) and the RNA-dependent cleavage of VP2/4 (13), the 3C protease is absolutely required for the cleavage of additional junction sites within the polyprotein (14,C16). In the mean time, EV71 3C reportedly interferes with the polyadenylation of sponsor cell RNA by digesting CstF-64, a critical host element for 3 pre-mRNA processing, suggesting a novel mechanism by which picornaviruses use 3Cpro to impair sponsor cell function (17). In addition, the 3C protease can also cleave several factors and regulators associated with cellular DNA-dependent RNA polymerases I, II, and III, such as the octamer-binding protein (OCT-1), TATA box-binding protein (TBP), cyclic AMP-responsive element-binding protein (CREB), transcription activator p53, histone H3, and DNA polymerase III (18,C21). The pivotal part of 3C protease in EV71 replication makes it a good target for antiviral finding (22). The crystal structure of unliganded EV71 3C protease showed that EV71 3C protease folded into two domains that are related to additional picornaviral 3C protease constructions (23). The complex constructions of EV71 mutants H133G, E71A, E71D with the inhibitor rupintrivir are similar to that of the unliganded protease structure (24). Lu et al. thoroughly characterized the 3C proteases from EV71 and CVA16 and reported a series of constructions of both enzymes in free, peptide-bound, or inhibitor-bound form (25). These findings provided exact molecular insights into the substrate acknowledgement and inhibition of 3C protease. Profiling of the EV71 3C protease substrate could not only provide in-depth knowledge of catalytic mechanism at a molecular level, which would facilitate the design of potent protease inhibitors, SL910102 but also lead to development of a reliable and powerful biochemical assay for screening. In 2008, Kuo et al. synthesized six dodecapeptide substrates derived from the EV71 protease cleavage site and one dodecapeptide substrate (TSAVLQSGFRKM) SL910102 from your severe acute respiratory syndrome coronavirus (SARS-CoV) protease autoprocessing site for biochemical characterization of the EV71 3C protease by determining their specificities using high-performance liquid chromatography (HPLC). The results showed that EV71 3C protease cleaved TSAVLQSGFRKM more efficiently than the additional six substrates (26). Eight peptides derived from CVA16 polyprotein and three peptides derived from EV71 polyprotein were also investigated for his or her susceptibilities to 3C cleavage via HPLC assay, and the peptide (IGNTIEALFQGPPKER) related to 2C-3A junction site of CVA16 could be efficiently processed by both proteases (= 8.37 M/min for EV71 and 10.72 M/min for CVA16) (25). However, the HPLC assay is limited to conveniently determine the substrate specification, and the technique of fluorescence resonance energy transfer (FRET) is commonly utilized for the preparation of fluorogenic substrates for biochemical characterization of proteases and protease inhibitor screening (27). A fluorogenic peptide, = 5.8 M; and of 7.1 10?4 M?1 min?1 (23), indicating that the peptide Dabcyl-RTATVQGPSLDFKE-Edans may not be the optimal substrate for EV71 protease inhibitor testing. The aim of this study was to biochemically characterize EV71 3C protease with an aim to develop a reliable and efficient assay for the screening of EV71 3C protease inhibitors. The substrate specificity was defined using a series of fluorogenic peptides mapped from your cleavage sites within the EV71 polyprotein. A peptide [P08, NMA-IEALFQGPPK(DNP)FR] with FRET organizations related to the cleavage site of the 2C-3A junction was identified to become the most efficiently cleaved by EV71 3C protease, having a kinetic constant of 11.8 0.82 mM?1 min?1. Compared with the substrates reported previously, P08 gave the highest signal-to-background ratio, which makes it an ideal substrate for assay development. The effects of pH, temp, and dimethyl sulfoxide (DMSO).