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Establishing isostructural metal substitution in metalloproteins using 1 H NMR, circular dichroism, and Fourier transform infrared spectroscopy
Author(s) -
Pountney Dean L.,
Henehan Colin J.,
Vasak Milan
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040815
Subject(s) - isostructural , rubredoxin , fourier transform infrared spectroscopy , chemistry , metalloprotein , crystallography , circular dichroism , infrared spectroscopy , spectroscopy , metal , nuclear magnetic resonance spectroscopy , analytical chemistry (journal) , amide , stereochemistry , organic chemistry , crystal structure , quantum mechanics , physics
Far‐UV CD, 1 H‐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 1 H‐NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.