Proteins in Motion
Elucidating proteins function at a level that allows for intelligent design and manipulation is essential in realization of their potential role in biomedical and industrial applications. It has become increasingly apparent though, that probing structures and functionalities under equilibrium conditions is not sufficient. Rather, many aspects of protein behavior and reactivity are rooted in protein dynamics. Thus, there is a growing effort to probe intermediate structures that occur transiently during the course of a proteins function in particular linked to the binding or release of a ligand or substrate. However, studies following the sequence of conformational changes triggered by the binding of sub-strate/ligand and the concomitant change in functional properties are inherently difficult because often the diffusion times are of the order of conformational relaxation times. This chapter describes methodologies for generating resonance Raman spectra from transient forms of hemoglobin under conditions that allow for the systematic exploration of conformational relaxation and functionality. Special consideration is given to Raman compatible protocols based on sol-gel encapsulation that allow for the preparation, trapping and temporal tuning of nonequilibrium population generated from either the addition or the removal of ligands/substrates.
Key WordsResonance Raman protein dynamics protein conformation ligand binding sol-gel hemoglobin.
- 7.Austin J., Jordan T., and Spiro T. (1993) Ultraviolet resonance Raman studies of proteins and related model compounds. In Biomolecular Spectroscopy Part A (Clark R. J. H. and Hester R. E., eds.), John Wiley and Sons, New York, pp. 55–127.Google Scholar
- 8.Kitagawa T. (1988) The heme protein structure and the iron histidine stretching mode. In Biological Application of Raman Spectroscopy, Vol. III (Spiro T. G., ed.), John Wiley & Sons, New York, pp. 97–131.Google Scholar
- 9.Rousseau D. L. and Friedman J. M. (1988) Transient and cryogenic studies of photodissociated hemoglobin and myoglobin. In Biological Applications of Raman Spectroscopy, Vol. III (Spiro T. G., ed.), John Wiley & Sons, New York, pp. 133–215.Google Scholar
- 16.Dave B. C., Miller J. M., Dunn B., Valentine J. S., and Zink J. I. (1997) Encapsulation of proteins in bulk and thin film sol-gel matrices. J. Sol Gel Sci. Technol. 8, 629–634.Google Scholar
- 21.Samuni U., Dantsker D., Khan I., Friedman A. J., Peterson E., and Friedman J. M. (2002) Spectroscopically and kinetically distinct conformational popula 300 tions of sol-gel encapsulated carbonmonoxy myoglobin: a comparison with hemoglobin. J. Biol. Chem. 25, 25.Google Scholar