Evaluating the conformation and binding interface of cap-binding proteins and complexes via ultraviolet photodissociation mass spectrometry
| Autor: | Patricia A. Murphy, Jennifer S. Brodbelt, Laura K. Mayberry, Karen S. Browning, John P. O'Brien |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular GTP' Stereochemistry Molecular Sequence Data Analytical chemistry Plasma protein binding Mass spectrometry Biochemistry Mass Spectrometry Protein Structure Secondary chemistry.chemical_compound Eukaryotic initiation factor 4F Homology modeling Amino Acid Sequence Cysteine Binding site Triticum Plant Proteins Binding Sites EIF4G Lysine Photodissociation food and beverages General Chemistry Recombinant Proteins Protein Structure Tertiary Dithiothreitol Eukaryotic Initiation Factor-4E chemistry Eukaryotic Initiation Factor-4F Guanosine Triphosphate Eukaryotic Initiation Factor-4G Sequence Alignment Protein Binding |
| Zdroj: | Journal of proteome research. 12(12) |
| ISSN: | 1535-3907 |
| Popis: | We report the structural analysis of cap-binding proteins using a chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating solvent accessibility of proteins. Our methodology utilized a chromogenic probe (NN) to probe the exposed amine residues of wheat eukaryotic translation initiation factor 4E (eIF4E), eIF4E in complex with a fragment of eIF4G ("mini-eIF4F"), eIF4E in complex with full length eIF4G, and the plant specific cap-binding protein, eIFiso4E. Structural changes of eIF4E in the absence and presence of excess dithiothreitol and in complex with a fragment of eIF4G or full-length eIF4G are mapped. The results indicate that there are particular lysine residues whose environment changes in the presence of dithiothreitol or eIF4G, suggesting that changes in the structure of eIF4E are occurring. On the basis of the crystal structure of wheat eIF4E and a constructed homology model of the structure for eIFiso4E, the reactivities of lysines in each protein are rationalized. Our results suggest that chemical probe/UVPD mass spectrometry can successfully predict dynamic structural changes in solution that are consistent with known crystal structures. Our findings reveal that the binding of m(7)GTP to eIF4E and eIFiso4E appears to be dependent on the redox state of a pair of cysteines near the m(7)GTP binding site. In addition, tertiary structural changes of eIF4E initiated by the formation of a complex containing a fragment of eIF4G and eIF4E were observed. |
| Databáze: | OpenAIRE |
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