Title
Multifaceted analyses of the interactions between human heart type fatty acid binding protein and its specific aptamers
Author
Ankana Kakoti, Pranab Goswami
Year
2016
Journal
Biochimica et Biophysica Acta (BBA) - General Subjects
Abstract
Aptamer-protein interaction studies have been mainly confined to dissociation constant (Kd) determination. A combinatorial approach involving limited proteolysis mass spectroscopy, molecular docking and CD studies is reported here to elucidate the specific interactions involved. To generate aptamers specific for human FABP3, SELEX was performed incorporating counter SELEX cycles against control FABPs and GST tag, followed by their characterization by EMSA, CD and SVD analysis. Based on computationally obtained aptamer-protein complex models, the interacting aptamer, and protein residues were predicted and supported by limited proteolysis experiments. Two aptamers N13 and N53 specific for human fatty acid binding protein (FABP3) were isolated with corresponding Kd of 0.0743 ± 0.0142 μM and 0.3337 ± 0.1485 μM for FABP3 interactions. Both aptamers possess stable B-DNA structures at salt concentration of 100 mM and pH range (6–9). The N13 aptamer led interaction involved 3 salt bridges and 2 hydrogen bonds, whereas N53 had 2 salt bridges with 8 hydrogen and 7 hydrophobic interactions. The aptamers generated are the first to be reported against human FABP3. The higher interaction footprint of N53 incited synergistic conformational changes in both N53 and FABP3 during interaction, leading to a decline in binding affinity in comparison to N13 which corroborated to the calculated Kd values.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Thermal stability, Ligand binding, Biochemistry