title: A sensitive method based on fluorescence-detected circular dichroism for protein local structure analysis.
abstract: We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca(2+) and/or interacting with binding peptides. Because FDCD appears to reflect the protein's local structure around the fluorophore, it may provide a useful means for "pinpoint analysis" of protein structures.
authors.affiliation: Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. tnehira@hiroshima-u.ac.jp
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Nehira
authors.forename: Kaoru
authors.initials: K
authors.lastname: Ishihara
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsuo
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Izumi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Ishida
mesh_terms: D000818:Animals; D001516:Bees; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008786:Metmyoglobin / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
chemical_list: D002147:Calmodulin; D005456:Fluorescent Dyes; D010455:Peptides; D008786:Metmyoglobin; D014357:Trypsin; D002118:Calcium