The thermal unfolding of a 40-residur helix-turn-helix subdomain of the P22 viral coat protein was investigated using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) with site-specific C-13 isotopic labelling. Helix-turn-helix is the simplest a-helical structural motif that combines both secondary and tertiary structural elements. The CD of individual helical fragments reveals that the P22 subdomain is stabilized by tertiary interhelical interactions. Overall the temperature-dependent CD and FTIR data can be described by a three-state process with a partially folded intermediate. However, the analysis of the site-specific C-13 IR signals reveals distinct unfolding thermodynamics for each jof the labeled sites. The thermodynamic parameters of the thermal unfolding of each of the labeled segements were obtained using singular value decomposition in combination with target transformation and global fitting. The P22 subdomain unfolds from the N-terminus toward the helical segments near the turn. Our results show that as few as two C-13 labeled residues can be detected in a 40 residue protein and provide local, site-specific structural information about protein unfolding, which is not resolved by standard, nonsite-specific spectroscopic probes.