In vitro translation and computational analyses of human profilin multimers.
Profilin is an ubiquitous intracellular G-actin and PIP2-binding protein that is a pan-allergen. Functional native human profilin multimers have recently been described, implicating regulatory roles in cell morphology, signaling and allergies. Considering the potential importance of profilin self-association in nature, multimerization was examined using cDNAs to human profilin I (P1) and II (P2) by employing a plasmid (pCITE2a+) for in vitro transcription/radiolabeled translation. Autoradiography of affinity column purified 35S-met-P1 or -P2 revealed predominant -14.8, 30 and 58 kDa bands that corresponded to parallel immunoblots using rabbit anti-human profilin antibodies. Reducing agents had no significant effect on the ratios of cpm associated with gel slices corresponding to 14.8/30/58 kDa profilin bands. Computer-based molecular modeling was used to further analyze a hypothetical structure for profilin multimers resistant to reducing agents. The crystalline P1 structure was downloaded and analyzed for likely sulphydryl bonding via the three integral cysteines (C16, C70, C127). All possible combinations of SH-bonds were investigated. The energetically most favorable dimer involved C16 + C127. 3-Dimensional conformation also revealed a protective pocket around the S-S bonds. The data show that, similar to native human and plant profilins, P1 and P2 cDNAs encode proteins that form fastidious multimers. A structure is proposed with relatively protected disulfide bridges between individual profilin moieties consistent with existing biochemical data.
Babich
Michael
M
Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois, USA. michaelb@immvarx.com
Foti
Lisa R P
LR
Wong
Linda
L
Pack
George R
GR
D001345:Autoradiography; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008958:Models, Molecular; D051304:Profilins / Q000096:biosynthesis* / Q000235:genetics*; D014176:Protein Biosynthesis; D016622:Silver Staining; D014158:Transcription, Genetic
D051304:Profilins