Use of CD and FT-IR to determine the secondary structure of purified proteins in the low-microgram range.
The spectroscopic characterization of protein secondary structure is often partially unreliable when samples are not extremely pure and abundant. This problem may be overcome by the combination of circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR). We used these methods to characterize the secondary structure of two proteins of neurobiological interest, calexcitin (CE) and the cellular isoform of prion protein (PrPC). Both proteins were purified with multiple chromatographic steps and were obtained in buffer with high purity (> 95%) and in low amount (approximately 2 micrograms). The samples were analyzed by circular dichroism (down to 184 or 182 nm), recovered, and deposited on films for infrared analysis. The spectral deconvolution from the two methods yielded secondary structures in good agreement with each other as well as with theoretical predictions based on amino acid sequence. The conformation of CE was found to be dependent on its concentration and on calcium binding. The secondary structure of cellular native PrP varied dramatically with the detergent used. In conclusion, the combination of CD and FT-IR analysis is suitable for the characterization of the conformational changes induced by ligand binding and/or by different solvent conditions when the protein of interest is only scarcely available. The methods used here provide valuable insights into the putative correlation between protein structure and activity.
Ascoli
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GA
Centro Studio CNR Macromolecole Stereordinate Otticamente Attive, Dipartimento di Chimica e Industriale, Università di Pisa, Italy.
Pergami
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P
Luu
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Alkon
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DL
Bramanti
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Bertucci
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C
Di Bari
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Salvadori
P
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D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D049832:Decapodiformes; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry; D005960:Glucosides / Q000737:chemistry; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D003902:Detergents; D005960:Glucosides; D011328:Prions; C018619:octyl-beta-D-glucoside; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C102751:cex-2 protein, C elegans; D002118:Calcium