The conformation of puroindoline-a (PIN-a), a protein extracted from wheat endosperm, in free-standing black films has been studied using confocal Raman spectroscopy. This protein is characterized by the presence in its sequence of a unique tryptophan (Trp)-rich domain and of five disulfide bridges stabilizing its three-dimensional structure. PIN-a is able to form free-standing films, which are very stable in time, because of its remarkable surface-active properties. These films become black in a few hours and are characterized by the presence of numerous aggregates. Their Raman spectra show major modifications or the PIN-a structure as compared to the solid form, such as the formation of beta-sheets or unordered structures, the modification of the environment of the Trp domain and, the conformation of disulfide bridges. These modifications are in agreement with an unfolding of the protein at the interfaces of the film and suggest that the Trp domain is involved in the aggregation. We have also studied the influence of increasing amounts of lysopalmitoylphosphatidylcholine (LPC) into the films. The direct observation of these mixed films shows that LPC inhibits the formation of PIN-a aggregates and that the conformation of PIN-a is strongly correlated to the LPC/PIN-a molar ratio. Raman spectroscopy also shows that PIN-a disturbs the highly organized arrangement of LPC molecules in the film.