Incorporation of the unnatural D-proline (P-D) stereo isomer into a polypeptide sequence is a typical strategy to encourage formation of beta-hairpin loops because natural sequences are often unstructured in solution. Using conformation-specific IR and UV spectroscopy of cold (approximate to 10 K) gas-phase ions, we probe the inherent conformational preferences of the P-D and P-L diastereomers in the protonated peptide [YAPAA+H](+), where only intramolecular interactions are possible. Consistent with the solution-phase studies, one of the conformers of [YADPAA+H](+) is folded into a charge-stabilized beta-hairpin turn. However, a second predominant conformer family containing two sequential gamma-turns is also identified, with similar energetic stability. A single conformational isomer of the P-L diastereomer, [YALPAA+H](+), is found and assigned to a structure that is not the anticipated "mirror image" beta-turn. Instead, the P-L stereocenter promotes a cis-alanine proline amide bond. The assigned structures contain clues that the preference of the P-D diastereomer to support a trans-amide bond and the proclivity of P-L for a cis-amide bond is sterically driven and can be reversed by substituting glycine for alanine in position 2, forming [YG(L)PAA+H](+). These results provide a basis for understanding the residue-specific and stereospecific alterations in the potential energy surface that underlie these changing preferences, providing insights to the origin of beta-hairpin formation.