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 (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–heme 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 ± 0.2 ns. Subsequently, the late, T-like I3 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 I3 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.

Photoinduced Structural Dynamics of Molecular Systems Mapped by Time-Resolved X-ray Methods

“…86, [332][333][334][335][336] Later, the same groups embarked in the study of dimeric hemoproteins, 337,338 with the aim to investigate cooperativity. Solution ps XRS is ideal when looking at conformational changes, large amplitude side chain motions and folding-unfolding processes and the above studies revealed several hitherto unknown details of the large scale protein dynamics.…”

Direct observation of bond formation in solution with femtosecond X-ray scattering

“…TRXSS is an effective method for probing photoinduced structural changes of molecules in solution and has been used to reveal the dynamics and mechanism of various molecular reaction systems ranging from small molecules [14][15][16][17] to biological macromolecules 18,19 . Although subpicosecond time resolution has been achieved in the case of X-ray 20-23 and electron [24][25][26] diffraction of solid samples and in X-ray absorption spectroscopy 27 of liquid samples, the temporal resolution of TRXSS based on synchrotron radiation 13 has been limited to only 100 ps, thus preventing the observation of ultrafast processes on the timescales of femtoseconds to picoseconds.…”

Anisotropic time-resolved solution X-ray scattering patterns from explicit-solvent molecular dynamics

“…1 This method was successfully applied to observe the structural dynamics of chemical reactions of small molecules in solution. [2][3][4][5][6][7][8] More recently, TR-WAXS was applied to track the conformational transitions of photo-active globular proteins [9][10][11][12][13][14][15][16] and proton pumps. [17][18][19][20] The structural interpretation of such TR-WAXS signals is challenging since, due to the orientational average inherent to solution X-ray scattering, WAXS patterns contain typically only 10-20 independent data points, 21 whereas proteins contain many more degrees of freedom.…”

“…11,12,15 Using such models, scattering curves for structural intermediates and low-resolution realspace electron densities of intermediates could be derived. 15 For a structural interpretation at the atomic level, TR-WAXS patterns have been calculated from structures determined by crystallography 9,10,14,16 or nuclear magnetic resonance (NMR). 13 In addition, Monte Carlo 14 and Molecular Dynamics (MD) simulations 4,5,10,16 have been used to refine structures a) jhub@gwdg.de; URL: http://cmb.bio.uni-goettingen.de/ against WAXS data.…”