Coupling Constant-pH and Accelerated MD Techniques to Explore the Conformational Landscape of a Mimetic Iron-binding Peptide

DIEGO SEBASTIAN VAZQUEZ; JAVIER SANTOS

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

SAB

One of the most common overlooked points in conventional molecular dynamics simulations is the change in the protonation state of titratable/ionisable residues during calculations. It is well known that protonation state has a very important role in protein structure, stability and function. Usually, the protonation state of each titratable residue is fixed along the simulation and is usually assigned by the standard ​ pKa value of the isolated residue in solution and the impact of fixed-settled protonation states would lead to an inaccurate or inadequate sampling of the native conformational ensemble of thebiomolecule. The high degree of conformational freedom in unstructured peptides is a challenging task. Furthermore, the interdependence between accessible conformations and protonation state is of particular interest given that these aspects could together influence the functional role of a specific sequence motif. In previous work, we grafted the putative iron-binding motif EExxED onto a foreign 16-Mer peptide scaffold. The designed peptide, called GRAP (from ​ GRAfted ​ Peptide ), consists of an acidic motif (EExxED) and was able to specifically bind Fe​ with a moderate affinity, an equimolar stoichiometry and the iron-peptide interaction was determined to be entropically driven. A set of mutant peptides of the acidic motif were designed and the metal-free conformational ensemble seems to be more unstructured than GRAP as judgedby their circular dichroism (CD) spectra between acidic and neutral pHs evidencing a clear protonation/conformation dependence. This scenariomakes exciting the study of the interdependence of the protonation state coupled to conformational changes and the ability to bind iron in a helical-restricted context with a simple peptide model. To address this issue, in the present work we coupled the constant-pH molecular simulation scheme with the unbiased conformational sampling aMDtechnique in explicit solvent for the GRAP and mutant peptides in a broad range of pHs along with the experimental determination of the pH-dependence of the total helical content by CD.