Establishing isostructural metal substitution in metalloproteins using 1H 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 micrograms 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.
Biochemistry Institute, University of Zürich, Switzerland.
D L
DL
Pountney
C J
CJ
Henehan
M
M
Vasák
D002942:Circular Dichroism; D003901:Desulfovibrio / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011522:Protons; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
D008667:Metalloproteins; D011522:Protons