The hairpin extension controls solvent access to the chromophore binding pocket in a bacterial phytochrome: a UV–vis absorption spectroscopy study

Abstract

Solvent access to the protein interior plays an important role in the function of many proteins. Phytochromes contain a specific structural feature, a hairpin extension that appears to relay structural information from the chromophore to the rest of the protein. The extension interacts with amino acids near the chromophore, and hence shields the chromophore from the surrounding solvent. We envision that the detachment of the extension from the protein surface allows solvent exchange reactions in the vicinity of the chromophore. This can facilitate for example, proton transfer processes between solvent and the protein interior. To test this hypothesis, the kinetics of the protonation state of the biliverdin chromophore from Deinococcus radiodurans bacteriophytchrome, and thus, the pH of the surrounding solution, is determined. The observed absorbance changes are related to the solvent access of the chromophore binding pocket, gated by the hairpin extension. We therefore propose a model with an “open” (solvent-exposed, deprotonation-active on a (sub)second time-scale) state and a “closed” (solvent-gated, deprotonation inactive) state, where the hairpin fluctuates slowly between these conformations thereby controlling the deprotonation process of the chromophore on a minute time scale. When the connection between the hairpin and the biliverdin surroundings is destabilized by a point mutation, the amplitude of the deprotonation phase increases considerably. In the absence of the extension, the chromophore deprotonates essentially without any “gating”. Hence, we introduce a straightforward method to study the stability and fluctuation of the phytochrome hairpin in its photostationary state. This approach can be extended to other chromophore-protein systems where absorption changes reflect dynamic processes of the protein.

The structural effect between the output module and chromophore-binding domain is a two-way street via the hairpin extension

“…2) meaning that due to uncoupling, the solvent accessibility to the chromophore is increased. This has been showed before in the photosensory module [51] and here also in full-length variants (Fig. 2).…”

“…In the population responsible for the fast phase, the tongue is in the "open" conformation and BV thus deprotonates rapidly. The slow phase corresponds to the kinetics of tongue opening from its closed state [51]. The use of stopped-flow spectrophotometry enabled measuring absorbance changes on sub-second time-scales (Fig.…”

“…Furthermore, the dynamic nature of the underlying signaling processes has been overlooked or its influence underestimated. To date, these shortcomings start to alleviate as structural information for Agp becomes available [53], enzymatic output for the DrBphP has been revealed [73], and the dynamic perspective of the protein subunits in their photostationary states has been reported [24,51].…”

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

“…We are happy to announce that today, this virtual volume unites 18 contributions by scientists from 17 countries (Canada, Czech republic, Finland, France, Germany, Hungary, India, Italy, Japan, Lebanon, Poland, Russia, Spain, Switzerland, Taiwan, UK, US) touching upon the wide variety of the subjects dealt with by Klaus at various times [25,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143].…”

“…The role of surprisingly slow protein dynamics is at the heart of a quasi-steady-state spectroscopic study: Rumfeldt et al used UV-Vis absorption spectroscopy on a seconds-tohours timescale to study solvent access to the chromophore binding pocket in bacterial phytochrome from Deinococcus radiodurans [130]. They propose a model where a protein hairpin fluctuates slowly between open and closed conformations, thereby controlling the deprotonation process of the chromophore on a minute time-scale.…”