Cross-linking with bifunctional reagents and its application to the study of the molecular symmetry and the arrangement of subunits in hexameric protein oligomers.
Cross-linking with a bifunctional reagent and subsequent SDS gel electrophoresis is a simple but effective method to study the symmetry and arrangement of subunits in oligomeric proteins. In this study, theoretical expressions for the description of cross-linking patterns were derived for protein homohexamers through extension of the method used for tetramers by Hajdu et al. (1976). The derived equations were used for the analysis of cross-linking by glutardialdehyde of four protein hexamers: beef liver glutamate dehydrogenase (GDH), jack bean urease, hemocyanin from the spiny lobster Panulirus pencillatus (PpHc), Escherichia coli glutamate decarboxylase (GDC) and for analysis of published data on the cross-linking of hexameric E. coli rho by dimethyl suberimidate. Best fit models showed that the subunits in the first four proteins are arranged according to D(3) symmetry in two layers, each subunit able to cross-link to three neighboring subunits for GDH and urease, or to four for PpHc and GDC. The findings indicate a dimer-of-trimers eclipsed arrangement of subunits for GDH and urease and a trimer-of-dimers staggered one for PpHc and GDC. In rho, the subunits are arranged according to D(3) symmetry in a trimer-of-dimers ring. The conclusions from cross-linking of GDH and GDC, PpHc and rho are consistent with results from X-ray crystal structure, those for urease with findings from electron microscopy.
Azem
Abdussalam
A
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Tsfadia
Yossi
Y
Hajouj
Omar
O
Shaked
Isabella
I
Daniel
Ezra
E
D000818:Animals; D032449:Canavalia / Q000201:enzymology; D002417:Cattle; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry; D005968:Glutamate Decarboxylase / Q000737:chemistry; D005969:Glutamate Dehydrogenase / Q000737:chemistry; D005976:Glutaral; D006433:Hemocyanins / Q000737:chemistry; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D033521:Palinuridae / Q000201:enzymology; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D013237:Stereoisomerism; D014510:Urease / Q000737:chemistry
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D021122:Protein Subunits; C534627:Rho protein, E coli; D006433:Hemocyanins; D005969:Glutamate Dehydrogenase; D014510:Urease; D005968:Glutamate Decarboxylase; D005976:Glutaral