Vibrational spectroscopy of proteins labeled with site-specific carbon-deuterium (CD) bonds is emerging as a powerful experimental technique for investigating protein structure, dynamics, and function. The C-D stretch mode makes an excellent probe because it is minimally perturbative and appears in an otherwise transparent region of the spectrum. The viability of Ì_å±-carbon deuterated bonds (alpha-C-D) as infrared (IR) probes of protein backbone dynamics was explored us- ing Ì_å±-carbon deuterated alanine (Ala-d1) as a convenient model system for a comparison of experiment, density functional theory (DFT), and combined quantum mechanical/molecular mechanical (QM/MM) simulations. In addition to the primary alpha-C-D absorption, the experimental spectrum contains three features resulting from Fermi resonances. DFT calculations confirmed the assignments and identified the lower frequency modes participating in the Fermi resonances. QM/MM simulations of the Ala-d1 line shape were in qualitative agreement with experiment. Two model dipeptide compounds, alpha-C-D labeled alanine dipeptide (Adp-d1) and alpha-C-D2 labeled glycine dipeptide (Gdp-d2), were used to validate the alpha-C-D vibrational probe as a reporter of local peptide conformation. These model com- pounds adopt structures that are analogous to the motifs found in larger peptides and proteins. The alpha-C-D2 frequencies of Gdp-d2 show the most substantial difference between its stable conformations: there is a 40.7 cmÌ¢è '1 maximum difference in the symmetric alpha-C-D2 stretch frequencies, and a 81.3 cmÌ¢è '1 maximum difference in the asymmetric alpha-C-D2 stretch frequencies. Moreover, the splitting between the symmetric and asymmetric alpha-C-D2 stretch frequencies of Gdp-d2 is remarkably sensitive to its conformation. The protonation state of titratable amino acid residues has profound effects on protein stability and function. Therefore, correctly determining the acid dissociation constant, pKa, of charged residues under physiological conditions is an important challenge. The ability of C-D vibrational probes to distinguish the protonation state of arginine, aspartic acid, glutamic acid, histidine, and lysine amino acid side chains was examined using DFT calculations. Lysine exhibited the largest C-D2 frequency shifts upon protonation, 44.9 cmÌ¢è '1 (symmetric stretch) and 69.5 cmÌ¢è '1 (asymmetric stretch). Furthermore, the predicted harmonic intensities of the C-D2 probe vibrations were extraordinarily sensitive to protonation state of the nearby acidic or basic group. Accounting for this dramatic change in intensity is essential to the interpretation of an IR absorption spectrum that contains the signature of both the neutral and charged states. Histidine is a particularly important contributor to protein stability and function because its side chain pKa is near physiological pH. The sensitivity of C-D vibrational frequencies to the protonation state of histidine dipeptide (Hdp) was investigated in aqueous solution using two-layered integrated molecular orbital and molecular mechanics (ONIOM) calculations. Solvating the labeled Hdp molecule produces an overall blue shift in the average C-D vibrational frequencies relative to the gas-phase. The beta-C-D2, delta-C-D, and epsilon-C-D vibrational probes all showed sensitivity to the histidine protonation state, with shifts of up to 40 cmÌ¢è '1 in the mean frequencies after deprotonation. The success of using C-D probes to distinguish protonation states in gas-phase and solvated dipeptides is encouraging for their application in proteins. Preliminary results calculating the epsilon-C-D frequency in protonated and deprotonated His12 in the protein ribonuclease S show good agreement to experimental results, although additional work is necessary to completely account for the spectral complexity in the experimental spectrum.