Structure-Correlation NMR Spectroscopy for Macromolecules Using Repeated Bidirectional Photoisomerization of Azobenzene

Control over macromolecular structure offers bright potentials for manipulation of macromolecular functions. We here present structure-correlation NMR spectroscopy to analyze the correlation between polymorphic macromolecular structures driven by photoisomerization of azobenzene. The structural conversion of azobenzene was induced within the mixing time of a NOESY experiment using a colored light source, and the reverse structural conversion was induced during the relaxation delay using a light source of another color. The correlation spectrum between <i>trans</i>- and <i>cis</i>-azobenzene was then obtained. To maximize the efficiency of the bidirectional photoisomerization of azobenzene-containing macromolecules, we developed a novel light-irradiation NMR sample tube and method for irradiating target molecules in an NMR radio frequency (rf) coil. When this sample tube was used for photoisomerization of an azobenzene derivative at a concentration of 0.2 mM, data collection with reasonable sensitivity applicable to macromolecules was achieved. We performed isomerization of an azobenzene-cross-linked peptide within the mixing time of a NOESY experiment that produced cross-peaks between helix and random-coil forms of the peptide. Thus, these results indicate that macromolecular structure manipulation can be incorporated into an NMR pulse sequence using an azobenzene derivative and irradiation with light of two types of wavelengths, providing a new method for structural analysis of metastable states of macromolecules

Nuclear Magnetic Resonance Detection of Hydrogen Bond Network in a Proton Pump Rhodopsin RxR and Its Alteration during the Cyclic Photoreaction

Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO– is strengthened and stabilizes the O-state

Segmental Isotope Labeling for Protein NMR Using Peptide Splicing

Segmental Isotope Labeling for Protein NMR Using Peptide Splicin

Specificity of Loop mediated isothermal amplification (LAMP) for <i>Mycobacterium ulcerans</i>.

<p>Conventional (upper, heat block) and pw-LAMP (lower, pocket warmer). Fluorescence image under the UV light are shown. Lanes; 1; <i>Mycobacterium ulcerans</i>, 2; <i>Mycobacterium marinum</i>, 3; <i>Mycobacterium shinsuense</i>, 4; <i>Mycobacterium tuberculosis</i>, 5; <i>Mycobacterium avium</i>, 6; <i>Mycobacterium intracellularie</i>, 7; <i>Mycobacterium kansasii</i>, 8; <i>Mycobacterium abscessus</i>, 9; <i>Mycobacterium chelonae</i>, 10; <i>Mycobacterium ulcerans</i>, 11; <i>Mycobacterium ulcerans</i> and 12; Jurkart cell line.</p

NMR Crystallographic Approach to Study the Variation of the Dynamics of Quinine and Its Quasienantiomer Quinidine

The structure and dynamics of quinine and its quasienantiomer quinidine were studied at the atomic resolution by measuring the chemical shift anisotropy (CSA) tensor and site-specific spin–lattice relaxation time. For quinine, there are three crystallographically independent molecules “a”, “b”, and “c” in an asymmetric unit since its 13C CP-MAS SSNMR spectrum features three distinct resonance peaks for certain carbon nuclei. The 13C assignments are fulfilled by DFT calculations. The experimental 13C isotropic chemical shifts well match the calculated values. These variations of isotropic chemical shift for three independent molecules are also observed by two-dimensional 13C–1H heteronuclear correlation spectroscopy (HETCOR) of quinine. The spin–lattice relaxation time, and the principal components of CSA parameters are also varied substantially for certain carbon nuclei of “a”, “b”, and “c” molecules. For quinidine, its 13C CP-MAS SSNMR spectrum is remarkably different from that of quinine despite, their almost identical solution NMR spectra. Furthermore, the remarkable change in the structure and dynamics of quasienantiomers are also observed including the steric effect of the substituent vinyl group, the variation of helical motifs, and the variation of the strength of the intermolecular hydrogen bonds. The variation of the structure and dynamics of quasienantiomers are thoroughly studied by solid-state NMR measurements. These types of studies will enrich the field of NMR crystallography

Comparison of IS<i>2404</i> PCR with pocket warmer LAMP for the detection of <i>Mycobacterium ulcerans</i> using different DNA templates.

*<p>Sensitivity as compared with IS<i>2404</i> PCR.</p><p>LAMP (Loop mediated isothermal amplification).</p

Detection of <i>Mycobacterium ulcerans</i> under ambient illumination.

<p>Tubes containing <i>M. ulcerans</i> DNA produced greenish fluorescence (tubes 1–4).</p

Comparison of IS<i>2404</i> PCR with conventional LAMP for the detection of <i>Mycobacterium ulcerans</i> using different DNA templates.

*<p>Sensitivity as compared with IS<i>2404</i> PCR</p><p>LAMP (Loop mediated isothermal amplification).</p

Identification of the N- and C-Terminal Substrate Binding Segments of Ferredoxin-NADP⁺ Reductase by NMR^†^,^@

Ferredoxin-NADP+ reductase (FNR) catalyzes the reduction of NADP+ through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP+- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP+ was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP+, respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites

Solution Structure of an RNA Duplex Including a C−U Base Pair^†^,^‡

The formation of the C−U base pair in a duplex was observed in solution by means of the temperature profile of 15N chemical shifts, and the precise geometry of the C−U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG]·r[CCUGCGUCG], it was found that a single C−U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C−U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C−U pair. The temperature profile of its 15N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C−U pair in the duplex