Use of 13C-labeled molecules in the conformational analysis of proteins by Ftir spectroscopy

DOIResolve DOI: http://doi.org/10.1007/978-94-011-0371-8_37
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TypeBook Chapter
Proceedings titleSpectroscopy of Biological Molecules : 6th European Conference on the Spectroscopy of Biological Molecules, 3–8 September 1995, Villeneuve d’Ascq, France
Conference6th European Conference on the Spectroscopy of Biological Molecules, September 3–8, 1995, Villeneuve d’Ascq, France
ISBN978-94-010-4166-9
978-94-011-0371-8
Pages8384; # of pages: 2
AbstractExtensive overlap of the diagnostic IR bands restricts the application of FTIR spectroscopy to the study of conformational changes when two proteins (peptides) form a complex. However, by mixing an isotopically carbon-13 labeled protein with an unlabeled protein or peptide, it is feasible to study their respective conformation-sensitive amide I bands separately [1]. While 15N labeling causes only subtle changes to the amide I band and to certain side chain absorptions, labeling of proteins with 13C results in a 50–55 cm-1 shift of the amide I band toward lower wavenumbers. We have used this strategy to probe the interaction of calmodulin, a calcium-binding protein (148 amino acids), with small synthetic peptides (22 amino acids) derived from the calmodulin-binding domain of three different target proteins [2]. Calmodulin was uniformly labeled (> 99%) with 13C in order to ensure the lack of any remaining intensity arising from calmodulin in the amide I region between 1640–1700 cm-1, a region in which the relatively weak amide I bands of the target peptides occur. An analysis of the IR spectra revealed that upon binding to calmodulin the conformation of the target peptides changes from an irregular structure to an a-helical conformation. In addition, a slight but perceptible pertubation of the conformation of calmodulin upon binding of the peptides was observed. The isotope-shifting strategy should be more generally applicable to the study of many other protein-peptide and protein-protein interactions, and should also prove useful for studying protein-nucleic acid interactions.
Publication date
PublisherSpringer Netherlands
LanguageEnglish
AffiliationNational Research Council Canada; Medical Devices
Peer reviewedNo
NRC number423
NPARC number9722737
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Record identifierb1573a94-428c-4131-bb71-dbe2bb561ec5
Record created2009-07-17
Record modified2016-11-28
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