Comparing Solution Structures of Amylin and CGRP by Nanosecond Laser-Pump Spectroscopy and Atomistic Simulations

Abstract

Amylin and calcitonin gene-related peptide (CGRP) are intrinsically disordered proteins, members of the calcitonin (Ct) peptide family. They are found with their respective receptors in different organs and carry out different functions. Amylin is involved in regulating glucose metabolism and is implicated in type II diabetes, while CGRP is a vasodilator involved in transmitting pain signals in the nervous system, and triggers migraine attacks. Amylin and CGRP share 47% sequence homology and are able to bind to each other's receptors and activate cell response. Such cross-reactivity is attributed to their possible structural similarity. Solution state NMR experiments show that both peptides are disordered and locally sample transient helical states close to the N-terminus. While such short-range structural properties have been compared, it is not clear whether the long-range properties are affected or not. Here we combine results from experiments, probing both long- and short-range properties of the two peptides, with results from replica exchange molecular dynamics (REMD) simulations. To measure a long-range property directly comparable to simulations, we use a nanosecond laser-pump spectroscopy technique based on tryptophan triplet quenching. This allows probing both the end-to-end distance and the rate of end-to-end contact formation in IDPs, without using prosthetic dyes. Because of the short length of our peptides and the high aggregation propensity of amylin, this information cannot be obtained using other techniques such as FRET. Our data show that both the secondary structure content and the end-to-end distance of the two peptides differ significantly, and that such differences are affected by electrostatic interactions. Both our experiments and REMD simulations indicate that long-range interactions (i.e. interactions between residues that are far away in the sequence), play a significant role in determining the peptide structural ensemble in solution.