title: New strategy for reversible modulation of protein activity through site-specific conjugation of small molecule and polymer.
abstract: A new strategy for accurate and reversible modulation of protein activity via simple conjugation of the sulfhydryl modifier and polymer with the introduced Cys residue in protein was developed in this study. With Escherichia coli inorganic pyrophosphatase (PPase) as a model protein, we used site-directed mutagenesis to generate a mutant PPase (PPC) with a substituted Cys residue at the specific Lys-148 site, which is within a conserved sequence near the active site and exposed to the surface of the PPC for chemical reaction. The site-specific conjugation of the mutated Cys residue in PPC with sulfhydryl modifier p-chloromercuribenzoate (PCMB) and pyridyl disulfide-functionalized poly(2-hydroxyethyl methacrylate) (pHEMA) resulted in obvious decrease or complete loss of the catalytic activity of PPC, due to the conformational change of PPC. Compared with the effect of small molecule modification (PCMB), the pHEMA conjugation led to greater inhibitory effect on protein activity due to the significant change of the tertiary structure of PPC after conjugation. Moreover, the protein activity can be restored to different extents by the treatment with different amount of reductive reagents, which can result in the dissociation between PPC and PCMB or pHEMA to recover the protein conformation. This study provides a new strategy for efficient control of protein activity at different levels by site-specific conjugation of a small molecule and polymer.
authors: The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University , 199 Ren'ai Road, Suzhou, 215123, P. R. China.
authors: Lei
authors: L
authors: Wang
authors: Lin
authors: L
authors: Yuan
authors: Hongwei
authors: H
authors: Wang
authors: Xiaoli
authors: X
authors: Liu
authors: Xinming
authors: X
authors: Li
authors: Hong
authors: H
authors: Chen
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008689:Methacrylates / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011102:Polyhydroxyethyl Methacrylate / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008689:Methacrylates; D011108:Polymers; D013438:Sulfhydryl Compounds; D011102:Polyhydroxyethyl Methacrylate; D020245:p-Chloromercuribenzoic Acid; C005044:hydroxyethyl methacrylate; D011755:Pyrophosphatases