IR Spectroscopic Discrimination between the Loop and -Helices and Determination of the Loop Diffusion Kinetics by T-jump Time-Resolved IR Spectroscopy for Cytochrome c

¹ Institute of Physics, CAS
² Institute of Physics, Chinese Academy of Sciences

^* To whom correspondence should be addressed. E-mail: yxweng{at}aphy.iphy.ac.cn.

Submitted on February 14, 2007
Revised on March 23, 2007
Accepted on 31 May 2007

	Abstract

The IR absorption of the amide I band for the loop structure may overlap with that of the -helices, which can lead to the mis-assignment of the protein secondary structures. 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 (cyt c ) in D₂O at a pD around 7.0. This small loop consists of residues 70 to 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^oC), well before the global unfolding of the secondary structure (Tm82^oC). This loop structural change is assigned as being triggered by the Met80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25^oC to 35 ^oC breaks the Fe-S bond between the Met80 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 3.6x10⁶ s^-1.

Key Words: Protein structure, alpha-helix, cytochrome c, loop, temperature-jump, time-resolved IR spectra