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

Abstract

Far-UV CD, '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 pg total protein) and, unlike either CD or '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.Keywords: amide I band; far-UV CD; FTIR; isostructural metal replacement; metal substitution; NMR; rubredoxin; secondary structure In order to study the structure of a metalloprotein, it is often necessary to replace the native metal ion, for example Zn, by another metal, such as Co, ]I3Cd, or 57Fe, the properties of which enable the structure of the protein to be probed using a certain spectroscopic technique, such as UV-visible absorption, NMR, Mossbauer, etc. The methods used to investigate metalbinding sites in proteins have been reviewed recently (Coleman, 1993; Maret & Vallee, 1993; Munck et al., 1993). Although structural alterations may occur due either to the presence of a nonnative metal ion or to the preparative treatment used in obtaining a metalloderivative, the preservation of the native protein conformation in the metalloderivative is usually just assumed.The objective of the present work is to establish a suitable and facile spectroscopic method for confirming experimentally that the polypeptide fold of a metalloprotein is preserved following metal substitution. We have evaluated the suitability of three established methods for examining the secondary structure compositions of proteins that use CD, Fourier transform infrared (FTIR), and 'H NMR spectroscopic techniques, respectively, for comparing the structures of a native and metal-substituted "

Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies

“…oleo orans [20]. CD, NMR and Fourier-transform IR spectroscopies have been used previously to demonstrate that cadmium-substituted D. gigas rubredoxin is isostructural with the native iron-containing rub- redoxin [30]. The similarity of the electronic absorption and CD spectra of the iron and cadmium forms of the C-terminal domain of Ps.…”

“…Rubredoxins have been studied extensively from the structural viewpoint. Three-dimensional structures with resolutions smaller than 1.5 A / have been reported [23][24][25][26][27], and NMR studies have been used to investigate metal ion ligation [28][29][30], contributions made by protein structure to the overall thermostability of rubredoxins [31] and solution structure [32,33]. However, despite this detailed structural knowledge, little is known about the function of rubredoxins in general.…”

Recombinant two-iron rubredoxin of Pseudomonas oleovorans: overexpression, purification and characterization by optical, CD and 113Cd NMR spectroscopies

“…viewpoint. Three-dimensional structures with resolutions better than 1.5 A H have been reported [9][10][11][12][13][14] and NMR studies have been utilized to investigate metal ion ligation [15,16], contributions made by protein structure to the overall thermostability of rubredoxins [17] and solution structure [18]. However, despite this detailed structural knowledge, little is known about the function of rubredoxins in general.…”

Silver‐induced conformational changes of polypeptides: a CD study

“…The direct CD measurements (θ, in mdeg) were converted to molar ellipticity, Δε (M −1 ·cm −1 ). Proportions of each secondary structure type were obtained by spectral deconvolution of the peptide CD spectrum as a linear sum of predetermined basis spectra, using the software on the instrument16. In addition, DICHROWEB (http://www.ogic.ca/projects/k2d2/) was also used for the deconvolution of spectra because it provides a user‐friendly interface to the existing programs and databases and enables a wide range of input formats and limits the need for pre‐analysis processing and conversion programs17.…”