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Construction of a Subnanosecond Time-Resolved, High-Resolution Ultraviolet Resonance Raman Measurement System and Its Application to Reveal the Dynamic Structures of Proteins

Volume 62, Number 1 (Jan. 2008) Page 30-37

Kubo, Minoru; Uchida, Takeshi; Nakashima, Satoru; Kitagawa, Teizo


A subnanosecond time-resolved ultraviolet (UV) resonance Raman system has been developed to study protein structural dynamics. The system is based on a 1 kHz Nd:YLF-pumped Ti:Sapphire regenerative amplifier with harmonic generation that can deliver visible (412, 440, 458, and 488 nm) and UV (206, 220, 229, and 244 nm) pulses. A subnanosecond (0.2 ns) tunable near-infrared pulse from a custom-made Ti:Sapphire oscillator is used to seed the regenerative amplifier. A narrow linewidth of the subnanosecond pulse offers the advantage of high resolution of UV resonance Raman spectra, which is critical to obtain site-specific information on protein structures. By combination with a 1 m single spectrograph equipped with a 3600 grooves/mm holographic grating and a custom-made prism prefilter, the present system achieves excellent spectral (<10 cm−1) and frequency (∼1 cm−1) resolutions with a relatively high temporal resolution (<0.5 ns). We also report the application of this system to two heme proteins, hemoglobin A and CooA, with the 440 nm pump and 220 nm probe wavelengths. For hemoglobin A, a structural change during the transition to the earliest intermediate upon CO photodissociation is successfully observed, specifically, nanosecond cleavage of the A-E interhelical hydrogen bonds within each subunit at Trpα14 and Trpβ15 residues. For CooA, on the other hand, rapid structural distortion (<0.5 ns) by CO photodissociation and nanosecond structural relaxation following CO geminate recombination are observed through the Raman bands of Phe and Trp residues located near the heme. These results demonstrate the high potential of this instrument to detect local protein motions subsequent to photoreactions in their active sites.