Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

B. Rusconi, A. C. Maranhao, J. P. Fuhrer, P. Krotee, S. H. Choi, F. Grun, T. Thireou, S. D. Dimitratos, D. F. Woods, O. Marinotti, M. F. Walter, E. Eliopoulos

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

Original languageEnglish
Pages (from-to)947-953
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1824
Issue number8
DOIs
StatePublished - Aug 2012

Keywords

  • 3D modeling
  • Anopheles gambiae
  • Circular dichroism spectroscopy
  • Fluorescence spectroscopy
  • Odorant Binding Protein
  • Site directed mutagenesis

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