Direct observation of myoglobin structural dynamics from 100 picoseconds to 1 microsecond with picosecond X-ray solution scatteringwz

Myoglobin (Mb) is a heme protein that carries small-molecule ligands such as O2, CO and NO in muscles, and can be considered as a subunit of hemoglobin, a paradigm protein for the study of allostery. Due to its small size, availability and photosensitivity of the heme–ligand bond, Mb has served as a prototypical model system for studying protein structural dynamics. Accordingly, structural dynamics of Mb have been intensively studied with various spectroscopic and structural tools. The ligands form covalent bonds with Fe of the heme group and can be photolyzed by visible light on the subpicosecond timescale. Upon the CO photolysis of MbCO, a small portion of the dissociated CO ligands geminately rebind to the heme, while the remainder travel to various pockets that can accommodate the ligand and eventually escape the protein matrix to the solvent. On a longer timescale, the vacant heme recovers the ligand via non-geminate recombination. To directly track the structural changes associated with the ligand migration and rebinding and capture structurally distinct intermediates, we used the pump-probe time-resolved X-ray solution scattering technique, where the time-dependent scattering of short X-ray pulses from a synchrotron are used to interrogate the structural dynamics of a liquid solution sample that is pumped with optical laser pulses in a pump-probe manner. Time-resolved X-ray solution scattering together with time-resolved X-ray crystallography, X-ray absorption spectroscopy and electron diffraction can provide direct structural information, and thus complements time-resolved optical spectroscopy in the analysis of solution-phase reaction mechanisms. Recently the time-resolved solution scattering technique has been applied to follow conformational changes in proteins with nanosecond and picosecond time resolution. Here, we show its application to another type of protein, Mb from equine heart, with picosecond time resolution. Time-resolved X-ray solution scattering data were measured at 14IDB beamline of Advanced Photon Source. The usual experimental protocol was followed. Specifically, equine heart MbCO solution (8 mM, pH 7.0, 0.1 M sodium phosphate) filled in a quartz capillary was excited by a B30 ps-long laser pulse at 532 nm to initiate the CO photodissociation, and a B100-ps-long single X-ray pulse was sent to the sample at a well-defined time delay with respect to the arrival of the laser pulse. The scattered X-rays were recorded in a CCD detector as a function of the time delay between the laser and X-ray pulses. Fig. 1A shows the difference X-ray scattering data measured for a wide time range spanning from 100 ps to 1 ms. The difference scattering curves, DS(q, t), were obtained by subtracting the scattering curve measured at 5 ms from the curves at various (positive) time delays, i.e., DS(q, t) = S(q, t) S(q, 5 ms). Oscillatory features are clear in the difference curves at all time delays. Most of the features in the difference signal develop within 100 ps, but some features still evolve afterwards. To magnify the subtle changes, double difference curves (DDS(q, t; tref) = DS(q, t) DS(q, tref)) were obtained between the difference curves at two different time delays using various reference time points (100 ps, 1 ns, 10 ns and 70 ns). For example, DDS(q, t; 100 ps) highlights the change occurring between t and 100 ps time delays. Close inspection of the timeresolved X-ray scattering data reveals four distinct transitions well separated in timescales. The first one occurs at 300–500 ps, the second at 1–10 ns, the third at 10–100 ns, the fourth at 100 ns–1 ms. Difference curves at representative time delays for each time region are compared in Fig. 1C. The data at 0 ps and 100 ps are identical within our experimental error if the data at 0 ps is doubled (as shown in Fig. 1A) to compensate for the delayed signal buildup due to half overlap between the pump and probe pulses at 0 ps. The agreement between the two data indicates that a significant degree of tertiary structural changes occurs faster than the time resolution of our experiment, resulting in the 0 ps signal with the same oscillatory features as in the 100 ps signal. As time goes on, the negative peak around q = 0.7 Å 1 (p3 region) grows in two steps on the timescales of 300–500 ps and 1–10 ns. The negative peak around q=0.35 Å 1 (p2 region) increases (i.e., becomes more negative) on the 1–10 ns timescale and then decreases (i.e., becomes less negative) in 10–100 ns. These changes can be more clearly seen in the double difference curves. For example, DDS(q, 10 ns; 1 ns) is negative whereas DDS(q, 100 ns; 10 ns) is positive around q = 0.35 Å . On the Center for Time-Resolved Diffraction, Department of Chemistry, Graduate School of Nanoscience & Technology (WCU), KAIST, Daejeon 305-701, Korea. E-mail: [email protected]; Fax: (+82) 42-350-2810; Tel: (+82) 42-350-2884 w This article is part of the ‘Emerging Investigators’ themed issue for ChemComm. z Electronic supplementary information (ESI) available: Data collection protocol, data processing and data analysis. See DOI: 10.1039/ c0cc01817a COMMUNICATION www.rsc.org/chemcomm | ChemComm