Potassium ion channels selectively permeate K⁺, as well as Rb⁺ and Cs⁺ to some degree, while excluding Na⁺ and Li⁺. Conformations of alkali metal complexes of Ac-Tyr-NHMe, a model peptide of the selectivity filter in a K⁺ channel, were previously found to correlate with the permeability of alkali metal ions to a K⁺ channel by cold ion trap infrared spectroscopy. With an additional temperature-controlled ion trap, we examined the conformations of the alkali metal complexes, allowing the ions to collide with a He buffer gas at different temperatures, prior to spectroscopic investigation. The conformational distribution of the K⁺–peptide complex shows the most significant variation with temperature, which suggests that this complex has more flexibility when complexed with K⁺ and suggests lower barrier heights than other metal–peptide complexes. The variability of the conformational distribution with temperature for the ions follows the same order of ion permeability of a K⁺ channel. This work demonstrates that the additional temperature-controlled ion trap is a powerful tool to explore the conformational landscape of flexible molecular systems.