Infrared spectroscopic discrimination between the loop and alpha-helices and determination of the loop diffusion kinetics by temperature-jump time-resolved infrared spectroscopy for cytochrome c.
The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).
Ye
Manping
M
Laboratory of Soft Matter Physics, Beijing National Laboratory of Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China.
Zhang
Qing-Li
QL
Li
Heng
H
Weng
Yu-Xiang
YX
Wang
Wei-Chi
WC
Qiu
Xiang-Gang
XG
D003198:Computer Simulation; D045304:Cytochromes c / Q000737:chemistry* / Q000648:ultrastructure*; D004058:Diffusion; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009038:Motion; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared / Q000379:methods*
D045304:Cytochromes c