Charge Patterning, Salt Screening and Denaturant Expansion in the CGRP Neuropeptide

Abstract

Calcitonin gene related peptide (CGRP) is a neuropeptide of the calcitonin peptide family, which acts as a vasodilator and is involved in the transmission of pain signals in the nervous system upon binding to the correspondent receptor. It also triggers migraine attacks, and is a major therapeutic target for the prevention of migraines. In physiological conditions, CGRP is intrinsically disordered and, therefore, the binding to its receptor (or to drugs) will depend strongly on the structural and dynamical properties of the disordered unbound state. Such properties can be affected in vivo by changes in salt concentration and pH. However, while some information is available on CGRP's sampling of local secondary structural elements, very little is known about its long-range (“tertiary”) structural and dynamical properties. Detecting such properties is challenging because CGRP has a low molecular weight and samples many different conformations on very fast time scales. We use a nanosecond laser-pump spectroscopy technique, based on tryptophan triplet quenching, which allows probing the end-to-end distance and the rate of end-to-end contact formation in IDPs. This provides similar information to FRET, but without the use of prosthetic dyes. Our data show that CGRP populates compact states in buffer, which are extremely sensitive to pH and salt concentration. We find that a change from pH 8 to pH 3 can induce a significant expansion of conformations due to the modulation of charge interactions, with a dramatic change of the corresponding salt screening effects. This suggests a key role of specific charged residues in CGRP. In addition, we find a “denaturant expansion” effect that depends on the nature of the denaturant. The observations can be rationalized in terms of polymer models where the polyelectrolyte/polyampholyte nature of the peptide is taken into account.