Protein modeling combined with spectroscopic techniques: An attractive quick alternative to obtain structural information
Abstract
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques.
This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H-4-H-5-loop revealed eight amino acids residues, namely besides the previously reported amino acids ASP(443), Lys(480), LyS(501), Gly(502) and Arg(544), also Glu(446), Phe(475) and Glu(482), which form the complete ATP recognition site.
Moreover, we have proved that a hydrogen bond between Arg(423) and Glu(472) supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro(489) is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding.
Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr(232) induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities.
Phosphorylation at Thr(232) induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr(232) on 14-3-3 binding properties.