A series of photoswitchable, alpha-helical peptides Were Studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with (CO)-C-13-O-18 at various Positions ill the sequence was employed to spectrally isolate particular backbone positions. We show that a single (CO)-C-13-O-18 label call give rise to two hands along the diagonal of the 2D-IR spectrum, one of which is from in amide group that is hydrogen-bonded internally, or to a solvent molecule, and the other From a non-hydrogen-bonded amide group. The photoswitch enabled examination of both the folded and Unfolded state of the helix. For most sites, Unfolding of the peptide caused a shift of intensity from the hydrogen-bonded peak to the non-hydrogen-bonded peak. The relative intensity of the two diagonal peaks gives all indication of the fraction of molecules hydrogen-bonded at a certain location along the sequence. As this fraction varies quite substantially along the helix, we Conclude that the helix is not Uniformly folded. Furthermore, the shift ill hydrogen bonding is much smaller than the change of helicity measured by CD spectroscopy, indicating that non-native hydrogen-bonded or mis-folded loops are formed in the unfolded ensemble.