Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump鈥揚robe X-ray Solution Scattering

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• 作者：Kyung Hwan Kim ; Srinivasan Muniyappan ; Key Young Oang ; Jong Goo Kim ; Shunsuke Nozawa ; Tokushi Sato ; Shin-ya Koshihara ; Robert Henning ; Irina Kosheleva ; Hosung Ki ; Youngmin Kim ; Tae Wu Kim ; Jeongho Kim ; Shin-ichi Adachi ; Hyotcherl Ihee
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：April 25, 2012
• 年：2012
• 卷：134
• 期：16
• 页码：7001-7008
• 全文大小：575K
• 年卷期：v.134,no.16(April 25, 2012)
• ISSN：1520-5126

文摘

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 (I₁, I₂, and I₃) 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鈥揾eme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I₁ intermediate is generated within 100 ps and transforms to the R-like I₂ intermediate with a time constant of 3.2 卤 0.2 ns. Subsequently, the late, T-like I₃ intermediate is formed via subunit rotation, a decrease in the heme鈥揾eme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 卤 120 ns for the fully photolyzed form and 5.6 卤 0.8 渭s for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I₃ intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme鈥揾eme 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.