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	Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
	Chu, LHM ; Choy, WY ; Tsai, SN ; Rao, ZH ; Ngai, SM
	2010-05-11 ; 2010-05-11
关键词	substrate specificity SARS-CoV protease MALDI-TOF mass spectrometry synthetic peptide PROTEINASE SEQUENCE DESIGN MICE Biochemistry & Molecular Biology
中文摘要	Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CL(pro)) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CL(pro). Six target amino acid positions flanking the SARS-CoV 3CL(pro) cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the "cartridge replacement" approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CL(pro). Susceptibility of each peptide substrate to SARS-CoV 3CL(pro) cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CL(pro)-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CLpro that is facing the P1 ' position is suggested to be occupied by acidic amino acids, thus the P1 ' position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or beta-branching amino acids in P3 and P2 ' positions may also hinder the binding of SARS-CoV 3CL(pro). This study generates a comprehensive overview of SARS-CoV 3CL(pro) substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CL(pro) inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.
语种	英语 ; 英语
出版者	COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT ; WOODBURY ; 500 SUNNYSIDE BLVD, WOODBURY, NY 11797-2924 USA
内容类型	期刊论文
源URL	[http://hdl.handle.net/123456789/26960]
专题	清华大学
推荐引用方式 GB/T 7714	Chu, LHM,Choy, WY,Tsai, SN,et al. Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J],2010, 2010.
APA	Chu, LHM,Choy, WY,Tsai, SN,Rao, ZH,&Ngai, SM.(2010).Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry..
MLA	Chu, LHM,et al."Rapid peptide-based screening on the substrate specificity of severe acute respiratory syndrome (SARS) coronavirus 3C-like protease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry".(2010).

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