Exposing the Nucleation Site of Alpha Helix Folding: A Joint Experimental and Simulation Study

Abstract

α-helices being the most common secondary structures found ubiquitously in proteins, have been studied extensively for elucidating its folding dynamics and mechanism. In addition, the interest in studying and arriving at a mechanistic understanding of the α-helix formation is motivated by the effort to design and develop individually folded, stable α-helices with novel biological functions and therapeutic utilities. The kinetics of this helix folding has been fundamentally described by a series of discrete steps where the nucleation event is the slowest and occurs first, followed by a faster elongation process. Even for a homo-polypeptide, this α-helical structure is inherently asymmetric, due to the specific alignment of the helical H-bonds between i and i+4 amino acid residues in the sequence. Thus, the key question remains whether the inherent asymmetry induces a directionality in the folding pathway; i.e. the productive nucleation event occurs randomly or at a specific location within the peptide sequence. To address this question, we have utilized crosslinking strategy to stabilize α-helical conformation in short alanine-based peptides to introduce specifically localized nucleation sites along the peptide chain. Laser induced temperature-jump (T-jump) infrared (IR) spectroscopy and molecular dynamics (MD) simulations have been then used on these pre-nucleated peptides to show that indeed the folding occurs faster when the helix is nucleated at the N-terminus rather than at the C-terminus of the peptides, giving a directionality to helix formation.