Analysis of the peptide helicity using an ensemble spectroscopic model with re-calibrated parameters

Abstract

AbstractThe α-helix is the fundamental building block of protein structure. Understanding the physical principles that determine how a specific amino acid sequence defines helicity is a key step towards elucidating the sequence-structure relationship. An established method for quantitation of helix content using circular dichroism (CD) relies on the linear spectroscopic model. In this model, the helix length-correction is not applied to each ensemble conformer individually, rather an average value is assumed for all conformers. Here we assess the validity of this approximation and introduce a more physically realistic ensemble-based analysis of the CD signal. We find that the linear model tends to underestimate peptide helicity, with the difference depending on the ensemble composition. Using a CD dataset covering a broad range of helicities, we re-calibrate spectroscopic parameters (helix and coil baselines) and determine helix-coil parameters for a set of alanine-rich peptides. Our results show that the ensemble model can leverage the small spectroscopic differences between peptide conformers, enabling it to extract more information from the experimental data. We show that some previously poorly-defined quantities, such as helix nucleation constant and heat capacity change associated with helix folding, can be reliably determined using ensemble model. Overall, the presented ensemble-based treatment of the CD signal, together with the re-calibrated values of the spectroscopic baseline parameters, provides a coherent framework for the analysis of the peptide helix content.Abstract Figure