Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays
| Autor: | S.H. Choi, Trias Thireou, J.P. Fuhrer, Spiros D. Dimitratos, Brigida Rusconi, F. Grun, Daniel F. Woods, A.C. Maranhao, Elias Eliopoulos, Osvaldo Marinotti, Pascal Krotee, Marika F. Walter |
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| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Circular dichroism Anopheles gambiae Molecular Sequence Data Biophysics Biology Ligands Receptors Odorant Biochemistry Article Analytical Chemistry 03 medical and health sciences Protein structure Anopheles Animals Point Mutation Amino Acid Sequence Binding site Site-directed mutagenesis Molecular Biology Peptide sequence 030304 developmental biology 0303 health sciences Binding Sites Point mutation Ligand binding assay Circular Dichroism 030302 biochemistry & molecular biology biology.organism_classification Molecular biology 3. Good health 1-Naphthylamine Mutagenesis Site-Directed Insect Proteins |
| Zdroj: | Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics; Vol 1824 |
| ISSN: | 0006-3002 |
| Popis: | 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 to 22%) 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. |
| Databáze: | OpenAIRE |
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