The Helical Alanine Controversy:  An (Ala)<sub>6</sub> Insertion Dramatically Increases Helicity

Employing chemical shift melts and hydrogen/deuterium exchange NMR techniques, we have determined the stabilization of the Trp-cage miniprotein due to multiple alanine insertions within the N-terminal α-helix. Alanine is shown to be uniquely helix-stabilizing and this stabilization is reflected in the global fold stability of the Trp-cage. The associated free energy change per alanine can be utilized to calculate the alanine propagation value. From the Lifson−Roig formulation, the calculated value (<i>w</i><sub>Ala</sub> = 1.6) is comparable to those obtained for short, solubilized, alanine-rich helices and is much larger than the values obtained by prior host−guest techniques or in N-terminally templated helices and peptides bearing long contiguous strings of alanines with no capping or solubilizing units present.