title: Direct observation of cooperative protein structural dynamics of homodimeric hemoglobin from 100 ps to 10 ms with pump-probe X-ray solution scattering.
abstract: Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. 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 picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam  Scapharca inaequivalvis  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 interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I(3) intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme-heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.
authors.affiliation: Center for Time-Resolved Diffraction, Department of Chemistry, Graduate School of Nanoscience & Technology (WCU), KAIST, Daejeon, 305-701, Republic of Korea.
authors.forename: Kyung Hwan
authors.initials: KH
authors.lastname: Kim
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Muniyappan
authors.forename: Key Young
authors.initials: KY
authors.lastname: Oang
authors.forename: Jong Goo
authors.initials: JG
authors.lastname: Kim
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Nozawa
authors.forename: Tokushi
authors.initials: T
authors.lastname: Sato
authors.forename: Shin-ya
authors.initials: SY
authors.lastname: Koshihara
authors.forename: Robert
authors.initials: R
authors.lastname: Henning
authors.forename: Irina
authors.initials: I
authors.lastname: Kosheleva
authors.forename: Hosung
authors.initials: H
authors.lastname: Ki
authors.forename: Youngmin
authors.initials: Y
authors.lastname: Kim
authors.forename: Tae Wu
authors.initials: TW
authors.lastname: Kim
authors.forename: Jeongho
authors.initials: J
authors.lastname: Kim
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Adachi
authors.forename: Hyotcherl
authors.initials: H
authors.lastname: Ihee
mesh_terms: D018360:Crystallography, X-Ray; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009010:Monte Carlo Method; D011487:Protein Conformation; D012542:Scattering, Radiation; D012996:Solutions; D014965:X-Rays
chemical_list: D006454:Hemoglobins; D012996:Solutions