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A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline.

ABSTRACT: UV-visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.

SUBMITTER: Sakaguchi M

PROVIDER: S-EPMC5356500 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Publications

A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline.

Sakaguchi Miyuki M Kimura Tetsunari T Nishida Takuma T Tosha Takehiko T Sugimoto Hiroshi H Yamaguchi Yoshihiro Y Yanagisawa Sachiko S Ueno Go G Murakami Hironori H Ago Hideo H Yamamoto Masaki M Ogura Takashi T Shiro Yoshitsugu Y Kubo Minoru M

Journal of synchrotron radiation 20160101 1

UV-visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic ana ...[more]

PMID: 26698082

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Project description:Electronic absorption spectra of proteins are primarily characterized over the ultraviolet region (185-320 nm) of the electromagnetic spectrum. While recent studies on peptide aggregates have revealed absorption beyond 350 nm, monomeric proteins lacking aromatic amino acids, disulphide bonds, and active site prosthetic groups are expected to remain optically silent beyond 250 nm. Here, in a joint theoretical and experimental investigation, we report the distinctive UV-Vis absorption spectrum between 250 nm [? = 7338 M-1 cm-1] and 800 nm [? = 501 M-1 cm-1] in a synthetic 67 residue protein (?3C), in monomeric form, devoid of aromatic amino acids. Systematic control studies with high concentration non-aromatic amino acid solutions revealed significant absorption beyond 250 nm for charged amino acids which constitute over 50% of the sequence composition in ?3C. Classical atomistic molecular dynamics (MD) simulations of ?3C reveal dynamic interactions between multiple charged sidechains of Lys and Glu residues present in ?3C. Time-dependent density functional theory calculations on charged amino acid residues sampled from the MD trajectories of ?3C reveal that the distinctive absorption features of ?3C may arise from two different types of charge transfer (CT) transitions involving spatially proximal Lys/Glu amino acids. Specifically, we show that the charged amino (NH3+)/carboxylate (COO-) groups of Lys/Glu sidechains act as electronic charge acceptors/donors for photoinduced electron transfer either from/to the polypeptide backbone or to each other. Further, the sensitivity of the CT spectra to close/far/intermediate range of encounters between sidechains of Lys/Glu owing to the three dimensional protein fold can create the long tail in the ?3C absorption profile between 300 and 800 nm. Finally, we experimentally demonstrate the sensitivity of ?3C absorption spectrum to temperature and pH-induced changes in protein structure. Taken together, our investigation significantly expands the pool of spectroscopically active biomolecular chromophores and adds an optical 250-800 nm spectral window, which we term ProCharTS (Protein Charge Transfer Spectra), for label free probes of biomolecular structure and dynamics.

Dataset Information

A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline.

Publications

A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline.

Similar Datasets

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