We used ultraviolet resonance Raman (UVRR) spectra to examine the spatial dependence and the thermodynamics of a-helix melting of an isotopically labeled a-helical, 21-residue, mainly alanine peptide. The peptide was synthesized with six natural abundance amino acids at the center and mainly perdeuterated residues elsewhere. C-alpha deuteration of a peptide bond decouples C-alpha-H bending from N-H bending, which significantly shifts the random coil conformation amide III band; this shift clearly resolves it from the amide III band of the nondeuterated peptide bonds. Analysis of the isotopically spectrally resolved amide III bands from the external and central peptide amide bonds show that the six central amide bonds have a higher a-helix melting temperature (similar to32 degreesC) than that of the exterior amide bonds (similar to5 degreesC).