Tracking the structural dynamics of homodimeric hemoglobin and BLUF photoreceptor Synechocystis PixD proteins using time-resolved X-ray solution scattering

Abstract

Studying protein conformational changes in native aqueous environment is a challenging task. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed combined spectroscopy technique to reveal the conformational dynamics of homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis. However, the time-resolved spectroscopy techniques do not provide the structural information of partially and fully photolysed HbI that are required to understand the allostry of heme proteins. To gain more knowledge about allostery of heme protein, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type HbI and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I(1), I(2), and I(3)) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme-heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss.The earliest, R-like I(1) intermediate is generated within 100 ps and transforms to the R-like I(2) intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I(3) intermediate is formed via subunit rotation, a decrease in the heme-heme distance, and substantial gain of interfac...