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

Abstract

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. Copyright © 1995 The Protein Society