Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination.

Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of (13)C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between C(α) and C(ß) of Y63 in the chromophore, the ß-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of ß-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.

NMR protein structure determination in living E. coli cells using nonlinear sampling.

The cell is a crowded environment in which proteins interact specifically with other proteins, nucleic acids, cofactors and ligands. Atomic resolution structural explanation of proteins functioning in this environment is a main goal of biochemical research. Recent improvements to nuclear magnetic resonance (NMR) hardware and methodology allow the measurement of high-resolution heteronuclear multidimensional NMR spectra of macromolecules in living cells (in-cell NMR). In this study, we describe a protocol for the stable isotope ((13)C, (15)N and (2)H) labeling and structure determination of proteins overexpressed in Escherichia coli cells exclusively on the basis of information obtained in living cells. The protocol combines the preparation of the protein in E. coli cells, the rapid measurement of the three-dimensional (3D) NMR spectra by nonlinear sampling of the indirectly acquired dimensions, structure calculation and structure refinement. Under favorable circumstances, this in-cell NMR approach can provide high-resolution 3D structures of proteins in living environments. The protocol has been used to solve the first 3D structure of a protein in living cells for the putative heavy metal-binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in E. coli cells. As no protein purification is necessary, a sample for in-cell NMR measurements can be obtained within 2-3 d. With the nonlinear sampling scheme, the duration of each 3D experiment can be reduced to 2-3 h. Once chemical shift assignments and NOESY peak lists have been prepared, structure calculation with the program CYANA and energy refinement can be completed in less than 1 h on a powerful computer system.

Protein structure determination in living cells by in-cell NMR spectroscopy.

Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

A (13)C-detection NMR approach for large glycoproteins.

NMR spectroscopy has great potential to provide us with information on structure and dynamics at atomic resolution of glycoproteins in solution. In larger glycoproteins, however, the detrimental fast (1)H transverse relaxation renders the conventional (1)H-detected NMR experiments difficult. (13)C direct detection potentially offers a valuable alternative to (1)H detection to overcome the fast T(2) relaxation. Here, we applied (13)C-detected NMR methods to observe the NMR signals of (13)C-labeled glycans attached to the Fc fragment of immunoglobulin G with a molecular mass of 56kDa. Spectral analysis revealed that a (13)C-detected (13)C-(13)C NOESY experiment is highly useful for spectral assignments of the glycans of large glycoproteins. This approach would be, in part, complementary to (13)C-(13)C TOCSY and (1)H-detection experiments.

Light-dependent regulation of structural flexibility in a photochromic fluorescent protein.

The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood, because the crystal structures of the bright state of the protein did not provide an answer to the mechanism of the photochromism, and structural determination of the dark state has been elusive. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. Light-induced changes in interactions between the chromophore and beta-barrel are responsible for switching between the two states. In the bright state, the apex of the chromophore tethers to the barrel by a hydrogen bond, and an imidazole ring protruding from the barrel stabilizes the plane of the chromophore. These interactions are disrupted by strong illumination with blue light, and the chromophore, together with a part of the beta-barrel, becomes flexible, leading to a nonradiative decay process.

Paramagnetic relaxation-based 19f MRI probe to detect protease activity.

A novel design principle for 19F MRI probes detecting protease activity was developed. This principle is based on 19F MRI signal quenching by the intramolecular paramagnetic effect from Gd3+. The intramolecular Gd3+ dramatically attenuated the 19F probe signal, and the paramagnetic effect was cancelled by the probe hydrolyzation by caspase-3. Using this probe, it was shown that the probe could detect caspase-3 activity spatially from a phantom image using 19F MRI.

Backbone 1H, 13C and 15N assignments of a 59 kDa Salmonella typhimurium periplasmic oligopeptide binding protein, OppA.

Salmonella typhimurium OppA is the periplasmic oligopeptide-binding protein. Backbone resonances of OppA(D419N) on its own were assigned for approximately 90% of residues. Missing residues are localised around the ligand-binding site, suggesting conformational flexibility in the unliganded state.