Protein changes on the ultrafast timescale
In phytochrome Agp2 ultrafast proton-transfer occurs from the chromophore to a hydrogen-bonded water network in the protein prior to photoisomerization. The altered hydrogen bonds persist after photoisomerization and trigger together with the new chromophore geometry the activated protein conformation.
Living organisms developed optimal tools to fulfill a number of functions. In cells, proteins help to synthesize and breakdown substrates and are essential for energy and signal conversion. Chromoproteins are activated by light and use light as energy source and information. Processes such as the growth and development of plants, their measurement of day-length and consequent timing of flowering are also controlled by chromoproteins.
Phytochrome (Phy) constitutes a well-known example for this class of proteins. Phy can exist in two different stable states, the so-called red (Pr) and far-red (Pfr) states. Phy can switch between both stable configurations by light absorption. Thereby, the bilin cofactor of Phy rapidly isomerizes upon light absorption on a picosecond time-scale. Concomitantly, a reaction cascade leads to overall protein conformational changes on the millisecond to second timescale.
The protein interacts with light via a bilin cofactor. The latter isomerizes and triggers structural rearrangements that propagate across the protein and ultimately activate the biological function. This conversion of light energy into a special activated structure, a process that occurs in all photoreceptors, is only partially understood. Spectroscopic methods are very sensitive to the local structural changes occurring at the bilin cofactor. This dynamics could be traced with high time resolution. However, information about the subsequent long-range structural dynamics of the protein is still scarce.
The Pfr form of phytochrome Agp2 is addressed here by transient absorption spectroscopy in the mid-IR. The mid-IR signal reports essentially about time-dependent bond dynamics. The spectral range around 1850 cm-1 can be unequivocally assigned to the protein, since no bilin signals interfere in this window. A very broad absorption band – or continuum – indicates transient formation of a proton-loaded hydrogen-bonded water network (HBWN). This protein feature builds up impulsively upon excitation of the chromophore. It decays with a time constant of 1.5 ps, which is concomitant with the decay of the electronic excited state of the bilin (Figure 1). Remarkably, isomerization of the chromophore takes place as the electronic excited state decays, implying that the protein response sets in already before the structural change of the chromophore.
This behavior may be understood with the help of quantum chemical calculations. These indicate that chromophore excitation brings about a strong charge redistribution, which modifies the electric field sensed by the surrounding protein. This facilitates deprotonation of the chromophore (excited-state proton transfer) and induces an ultrafast protein response. In detail, the leaving proton is transferred to hydrogen-bonded water network (HBWN) consisting of polar amino acids, such as histidine, propionic side chains of the chromophore and protein bound water molecules. Consistent with this explanation, slower dynamics and lower transient concentration of the proton-loaded HBWN are observed upon mutation of the involved histidine residue or by decreasing the pH of the medium.
The spectral continuum in Figure 1 results from a proton continuously switching bonds among water molecules and the groups building up the HBWN. On a longer timescale, the electronic excited state decays, the original electric field is reestablished, and the leaving proton shifts from the HBWN back to the bilin. The continuum band decays concurrently.
The question that arises is whether the transient formation of a proton-loaded HBWN results in a long-lasting structural change in the protein, relevant to the biological function. Indeed, ultrafast changes of the HBWN around the propionic side chain C survive the photoisomerization, which develops further on a longer time-scale. The propionic side chains C is thought to be crucial for developing the activated protein conformation. Thus, one concludes that, upon optical excitation and prior to isomerization, protein structural changes relevant for downstream processes occur. These changes are driven by an ultrafast change of the electric field induced at earliest time by optical excitation of the bilin chromophore.
These new findings show that biologically relevant structural changes in photoreceptors can be launched by the ultrafast modulation of the local electric field resulting from chromophore excitation.