Photocleavage of lysozyme by cobalt(III) complexes.
Photochemical reagents that cleave proteins at specific sites (photoproteases) are useful for studying protein structure and protein-ligand interactions. PolyammineCo(III) complexes are tested here as photochemical probes to cleave proteins. Irradiation of a mixture of lysozyme, a model protein, and polyammineCo(III) complexes resulted in the facile cleavage of the peptide backbone. Photocleavage yielded two fragments of molecular weights 10.6 and 3.7 kDa, and these masses sum to the molecular mass of lysozyme (14.3 kDa). No cleavage was detected in the absence of the metal complex, in the dark, or upon irradiation at wavelengths of >420 nm. The photocleavage yield increased with irradiation time and with the concentrations of the metal complex and the protein. N-terminal sequencing of the 10.6 kDa fragment indicated residues that are identical to the N-terminus of lysozyme, and sequencing of the 3.7 kDa fragment indicated Val-Ala-Trp-Arg, an internal sequence of lysozyme. From the known primary sequence of lysozyme and the sequencing data, the cleavage site was assigned to Trp108-Val109. Molecular modeling indicates that the observed cleavage site is within few angstroms from the proposed metal binding site at Glu35-Asp52. This is the first report of the successful photocleavage of proteins, with high selectivity, by transition metal complexes. This novel observation can facilitate the rational design of transition metal complexes for the photochemical footprinting of metal binding sites on proteins.
Kumar
Challa V
CV
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, USA. Challa.Kumar@Uconn.edu
Thota
Jyotsna
J
D001665:Binding Sites; D002412:Cations; D003035:Cobalt / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism*; D009942:Organometallic Compounds / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D010777:Photochemistry; D010782:Photolysis; D013997:Time Factors
D002412:Cations; D009942:Organometallic Compounds; D010455:Peptides; D003035:Cobalt; D009113:Muramidase; D010447:Peptide Hydrolases