Molecular Dynamics Ensemble Refinement of Intrinsically Disordered Peptides According to Deconvoluted Spectra from Circular Dichroism

Jacob C Ezerski,Pengzhi Zhang,Nathaniel C Jennings,M Neal Waxham,Margaret S Cheung

doi:10.1016/j.bpj.2020.02.015

Abstract

We have developed a computational method of atomistically refining the structural ensemble of intrinsically disordered peptides (IDPs) facilitated by experimental measurements using circular dichroism spectroscopy (CD). A major challenge surrounding this approach stems from the deconvolution of experimental CD spectra into secondary structure features of the IDP ensemble. Currently available algorithms for CD deconvolution were designed to analyze the spectra of proteins with stable secondary structures. Herein, our work aims to minimize any bias from the peptide deconvolution analysis by implementing a non-negative linear least-squares fitting algorithm in conjunction with a CD reference data set that contains soluble and denatured proteins (SDP48). The non-negative linear least-squares method yields the best results for deconvolution of proteins with higher disordered content than currently available methods, according to a validation analysis of a set of protein spectra with Protein Data Bank entries. We subsequently used this analysis to deconvolute our experimental CD data to refine our computational model of the peptide secondary structure ensemble produced by all-atom molecular dynamics simulations with implicit solvent. We applied this approach to determine the ensemble structures of a set of short IDPs, that mimic the calmodulin binding domain of calcium/calmodulin-dependent protein kinase II and its 1-amino-acid and 3-amino-acid mutants. Our study offers a, to our knowledge, novel way to solve the ensemble secondary structures of IDPs in solution, which is important to advance the understanding of their roles in regulating signaling pathways through the formation of complexes with multiple partners.

Highlights

Disordered proteins/peptides (IDPs) are a category of proteins that possess a poorly defined equilibrium structure; they sample an ensemble of weakly ordered and unordered structures in solution [1,2,3,4,5]
With our combined approach of circular dichroism spectroscopy (CD) experiments and molecular dynamics (MD) simulations, we have unexpectedly discovered that the increase of secondary structures in a revealing peptide mutant (AAA) was due to the formation of a b-hairpin conformation, which we speculate is the mechanism behind changes in the encounter rate for this set of Intrinsically disordered proteins/peptides (IDPs)
CD spectra indicate a distinct secondary structure shift between RRK and a particularly revealing peptide mutant (AAA) The CD spectra presented in Fig. 1 show the average secondary structure ensembles of RRK, RAK, and AAA peptides: three peptides of identical length exhibiting significantly different binding kinetics with CaM [41]

Summary

Introduction

Disordered proteins/peptides (IDPs) are a category of proteins that possess a poorly defined equilibrium structure; they sample an ensemble of weakly ordered and unordered structures in solution [1,2,3,4,5]. A distinguishing feature of IDPs is that they do not adhere to the classical structure-function paradigm and typically form stable secondary or tertiary structures only upon binding to target proteins [12,13]. The lack of stable structures in the ensemble of unbound state [14,15,16,17] enables binding to multiple targets on demand while maintaining a degree of selectivity and specificity because of their polymorphic properties [18]. Multiple binding pathways exist between a given IDP and its protein targets [19]. IDPs are susceptible to post-translational modifications [20,21,22,23]

Methods

Results

Discussion

Conclusion