33] Monitoring of protein conformation changes during photocycle

Abstract

This chapter discusses the monitoring of protein conformation changes during photocycle. The absorption of light by the light-adapted purple membrane protein results in a photochemical reaction cycle through a series of intermediates. Accompanying this cycle is the pumping of protons across the membrane. To study this relationship in terms of possible protein conformation changes, the use of low-temperature ultraviolet (320–245 nm) absorption spectroscopy has proved valuable. At specific low temperatures, certain intermediates of the bacteriorhodopsin (BR) photocycle can be trapped or prevented from thermally converting to the next intermediate in the sequence. This method offers a number of distinct advantages in the study of protein changes in the photocycle intermediates. First, relatively large percentages of the pigment can be converted to the first three intermediates (K, L, and M). Samples can be prepared with 28% K, 65% L, or 100% M. In addition, the only other species found in significant amounts in these pigment mixtures is BR. This greatly simplifies analysis of the spectra of the intermediate.