Establishing isostructural metal substitution in metalloproteins using ¹ H NMR, circular dichroism, and Fourier transform infrared spectroscopy

Far‐UV CD, ¹H‐NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well‐characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 μg total protein) and, unlike either CD or ¹H‐NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.