In vitro translation and computational analyses of human profilin multimers

Michael Babich, Lisa R P Foti, Linda Wong, George R. Pack

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)39-43
Number of pages5
JournalProceedings of the Western Pharmacology Society
Volume48
StatePublished - 2005

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Profilins
Reducing Agents
Population Groups
Complementary DNA
Molecular Computers
In Vitro Techniques
Microfilament Proteins
Autoradiography
Disulfides
Allergens
Cysteine
Actins
Hypersensitivity
Plasmids
Gels
Rabbits

ASJC Scopus subject areas

  • Pharmacology

Cite this

In vitro translation and computational analyses of human profilin multimers. / Babich, Michael; Foti, Lisa R P; Wong, Linda; Pack, George R.

In: Proceedings of the Western Pharmacology Society, Vol. 48, 2005, p. 39-43.

Research output: Contribution to journalArticle

Babich, Michael ; Foti, Lisa R P ; Wong, Linda ; Pack, George R. / In vitro translation and computational analyses of human profilin multimers. In: Proceedings of the Western Pharmacology Society. 2005 ; Vol. 48. pp. 39-43.
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