Excimer fluorescence as a tool for monitoring protein domain dynamics applied to actin conformation changes based on circulary polarized fluorescence spectroscopy.
Fluorescence-detected circular dichroism (FDCD) was introduced into the study of protein conformation changes. Actin was used as a model protein which undergoes dynamic conformation changes as it polymerizes. Actin labeled with N-(1-pyrene)iodoacetamide (PIA) showed monomer fluorescence peak at 386 and 410 nm, and excimer fluorescence peak at around 480 nm. Excimer was formed by PIA-dimers labeled to different sites of amino acid residues. New information concerned with actin structural changes were monitored by fluorescence emission spectra excited with left- and right-circulary polarized light at 355 nm. FDCD intensities were shown as the difference in the fluorescence emission DeltaF, where DeltaF=(F (L)-F (R))/(F (L)+F (R)) denoting F (L) and F (R) as emissions obtained by excitation with left- and right-circulary polarized light. When solvent conditions of PIA-actin were changed by addition of NaCl, TFE, or ATP, DeltaF showed sensitive responses to these compounds. From the analysis of DeltaF (M) and DeltaF (E) which represent the peaks of DeltaF at the monomer- and excimer-emission band, the information concerned with the actin intrastructural changes were obtained. This method based on monitoring the excimer fluorescence with FDCD could be used for other proteins to extract finer structural changes that cannot be detected by the normal fluorescence spectroscopy.
Ikkai
T
T
Aichi Prefectural University of Fine Arts, Sagamine 1-1, Nagakute, Aichi 480-1194, Japan. ikkai@mail.aichi-fam-u.ac.jp
Arii
T
T
Shimada
K
K
D000199:Actins / Q000737:chemistry*; D000477:Alkylating Agents / Q000737:chemistry*; D002942:Circular Dichroism / Q000379:methods*; D005453:Fluorescence; D007460:Iodoacetamide / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000199:Actins; D000477:Alkylating Agents; D007460:Iodoacetamide