Abstract
Significance
Two-dimensional infrared absorption (2D-IR) spectroscopy is severely limited in its application to larger proteins due to broad and overlapping signals in the amide I region. Here, we overcome this limitation and isolate couplings between pairwise two single-molecular groups in the biotechnologically relevant phytochrome Agp1 (510 aa) by calculating light-induced difference spectra. In phytochromes, the photoactivation of a cofactor with a relatively small structural change triggers a large-scale refolding of big parts of the protein, but the mechanism of that interaction is not understood. We observe cross-peaks in the 2D-IR spectra that are directly related to the changing dipole coupling between the cofactor and the part of the protein that refolds, suggesting that both sites stabilize each other mutually.
Abstract
Phytochromes are ubiquitous photoreceptor proteins that undergo a significant refolding of secondary structure in response to initial photoisomerization of the chromophoric group. This process is important for the signal transduction through the protein and thus its regulatory function in different organisms. Here, we employ two-dimensional infrared absorption (2D-IR) spectroscopy, an ultrafast spectroscopic technique that is sensitive to vibrational couplings, to study the photoreaction of bacterial phytochrome Agp1. By calculating difference spectra with respect to the photoactivation, we are able to isolate sharp difference cross-peaks that report on local changes in vibrational couplings between different sites of the chromophore and the protein. These results indicate inter alia that a dipole coupling between the chromophore and the so-called tongue region plays a role in stabilizing the protein in the light-activated state.