Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography

Abstract

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-A resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100-300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (< or =3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function.

Fig. 1.

Structural changes in YQR-Mb 316 ns after photolysis. (a) Overlay of (F_light – F_dark) difference electron density maps on the YQR-MbCO model (red, negative; green, positive; contoured at 3.5σ, where σ is the standard deviation of the electron density difference). Most changes are clustered on the distal (upper) side of the heme. (b) Overview of the modeled structure of YQR-MbCO (red) and YQR-Mb* (green). The heme and several key residues are rendered as balls and sticks, the CO is rendered as space filling, and the rest of the protein is rendered as ribbon.

Fig. 2.

Detailed view of the time-dependent electron density differences (red, negative; green, positive) overlaid on the models of YQR-MbCO (yellow) and YQR-Mb* (blue). (a) Heme vicinity: the disappearance of bound CO, tilting of the heme, swinging motion of Tyr-29(B10), and migration of photolyzed CO to the Xe4 docking site (orange stick) are fully developed by 3 ns (contoured to 3.0σ); by 316 ns, the photolyzed CO has reached the Xe1 docking site (blue stick). (b) Concerted motion of Asp-60(E3), Phe-46(CD4), and Wat56, reaching a maximum amplitude by 316 ns (contoured to 3.0σ). (c) Overview of structural changes taking place around the E-helix, CD-turn, and F-helix (contoured to 4.0σ).

Fig. 3.

Time dependence of difference electron density for key structural features. The numerical values reflect the integral of the positive electron density beyond 3.5σ (negative density for CO hole) corrected for variations in photolysis yield. The error bars at 32-ns delay correspond to twice the rms deviation among the four data sets collected at this delay. (a) Key features that appear promptly. (b) Key residues whose displacement attains a maximum amplitude at ≈316 ns. (c) Secondary structures whose overall displacement also attains a maximum amplitude at ≈316 ns. The average integrated density per residue is shown; the occupation by CO of the Xe4 and Xe1 docking sites is also reported by displaying the total integrated density divided by 10 for scaling purposes.