DENSITOMETRIC ANALYSIS OF SYNCHROTRON XRAY FOOTPRINTING AUTORADIOGRAMS

Project: Research project

Project Details

Description

Understanding the folding kinetics of proteins is vital in revealing their biological functions and structures. Spectroscopic methods used to date have been quite helpful in characterizing transient-intermediate structures of proteins, but new methods to investigate tertiary structure changes are needed. Synchrotron x-ray footprinting techniques enable millisecond time-resolved structural changes in nucleic acid conformation and protein-nucleic acid complexes to be studied. This approach in conjunction with mass spectrometric sequencing methods is being used to study folding kinetics of cytochrome c. Hydroxyl radicals generated by x-ray radiolysis of water induce detectable amino acid modifications of cytochrome c within 4 milliseconds. Deconvoluted electrospray ionization mass spectra reveal that the prominent modification results in ions at +16 u intervals above the molecular weight of unmodified protein. Tryptic peptides of the modified protein were isolated and the modification quantitated. Although tandem experiments have not been completed, it was presumed that the following residues were modified based on our peptide results. F10, F36, F46, Y67, Y74, F82, and M80 were modified after 50 ms of exposure, while Y97 showed no modifications. The modification extents are correlated with the solvent accessibility residue map of cytochrome c.
StatusFinished
Effective start/end date10/1/959/30/02

Funding

  • National Center for Research Resources

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  • ALBERT EINSTEIN CENTER FOR SYNCHROTRON BIOSCIENCES

    Blasie, K. J., Chance, B., Chance, M. R., Almo, S., Anderson, R., Anni, H., Brenowitz, M. D., Burke, J., Chen, T., Crothers, D., Dauter, Z., Davies, D., Downard, K. M., Fisher, T., Franzen, S., Goldsmith, S., Heiney, P. A., Holmes, K. C., Hurley, J., Hurley, J., Ji, X., Kovacs, K., Kravtsov, I., Lee, C. C., Maleknia, S. D., Marinkovic, N., Maroney, M. J., Maroney, M., Miller, L. M., Morris, A., Pedersen, L., Penner-Hahn, J. E., Powers, L. S., Pyle, A. M., Que, L., Que Lawrence, J., Sagi, I., Scarrow, R. C., Scheuring, E., Seeman, N. C., Silva, A., Snyder, G., Sonenberg, N., Sperber, E., Studier, B., Sullivan, M. D., Sun, P., Toomey, J. R., Tran, A., Vanamee, E. S., Wlodawer, A., Yan, H., Bubacco, L., Caffrey, M., Chance, M. R., Kallenbach, N., Sharpe, L., Sinclair, R., Woodson, S., Xie, A. & Yue, K.

    8/1/858/31/03

    Project: Research project