Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Breland Crudup - Tougaloo College
Co-Author(s): Manliang Feng, Tougaloo College Tougaloo, MS
Electron transfer is an essential part of many biological processes. Effects of distance and static structures on electron-transfer among proteins have been studied extensively. Recent computational studies have suggested that protein motion is coupled to electron-transfer. However, few experimental results have been reported mainly because most spectroscopic methods which are the major means for studying molecular structure only look at the static structure. Moreover, protein motion and electron transfer take place in milliseconds to femtoseconds time scale. It is not possibleto study the dynamic structures ofthe protein due to current technical limitations. Our hypothesis is that protein motion involves two or more structural sub-states of different energies. The distribution of these sub-states is temperature dependent. Therefore, variable temperature spectroscopic method could identify and characterize the sub-structure involved in protein motion leading to better understanding of the electron-transfer mechanism. In this research, we use Raman spectroscopicand electrochemical methods to study the dynamic structure of Amicyanin, an electron-transfer mediator in a three-member redox complex involving methylamine dehydrogenase (MADH) and cytochrome c-551i in Paracoccusdenitrificans.Amicyanin was expressed and isolated in this laboratory from Escherichia coli BL21 cells. Purity of the protein was assessed by UV-Vis spectroscopy and SDS-PAGE. Resonance Raman spectra of amicyanin was measured with an Ocean Optics QE-65 Raman Spectrometer with an excitation wavelength of 532 nm. Calibration of the frequency was performed with cyclohexane standards. Resonance Raman spectrum of Amicyanin reveals 3 intense bands at 377, 392 and 430 cm-1 at 277 K. These peaks were attributed to the fundamental S(sys)-Cu stretch as well as modes due to coupling of internal ligand vibrational modes with the Cu-S stretch. Raman bands at 377 and 392 cm-1 exhibit a 2-4 cm-1 shift to higher frequencies at 77K with a narrowed band width. This result implies the copper center has a constant breathing motion causing changes of length and orientation of Cu-S bonds. At lower temperature, the protein motion is suppressed and the copper center assumes a structure with shorter Cu-S distance. Cyclic voltammetric (CV) study of Amicyanin adsorbed a modified gold electrode (where protein motion is inhibited) showed a Ep of 150 mV at 100mV/s scan rate. Simulation of the CV data suggests an electron transfer rate much low that that measured in solution. Current study indicated that variable temperature Raman spectroscopy could be an effective way to study sub-structures in protein motion and their roles in electron transfer. Future works will focuson Amicyanin in complex with MADH and cytochrome c551i as well as that with MauG to further elucidate the role of protein motion to electron-transfer in real biological systems.
Funder Acknowledgement(s): This work is supported by NSF Research Initiation Award under HBCU-UP program (Award number: 1505446). Author thanks Jackson Heart Study for travel support.
Faculty Advisor: Manliang Feng, email@example.com
Role: I did protein preparation and UV-Vis, Raman Spectroscopy, and electrochemistry.