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 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.

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 ␤-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 ␤-barrel, becomes flexible, leading to a nonradiative decay process.crystal structure ͉ NMR ͉ photochromism

Endohedral metallofullerenes have attracted special interest as spherical molecules with novel properties that are not expected from empty fullerenes.['] The higher fullerenes can even encapsulate two metal atoms inside the carbon cages to form soluble and relatively air-stable endohedral dimetallofullerenes. Because of the difficulty in producing pure samples in large quantities, experimental characterization of these species has been hindered. Important progress was marked by the successful isolation and purification of endohedral dimetallofullerenes such as Sc2 (iC,,12] and La,@C,,[31 in macroscopic quantities. This has made it possible to investigate redox propertiesr3] and reactivity.14] STM''] and TEMI61 studies were also carried out in an attempt to confirm that two metal atoms are inside the fullerene cages.Since there are many isomers for higher fullerenes, determination of cage structures and symmetries has long been of fundamental interest in disclosing the mechanism of growth. This has been a recent subject of theoretical14. ' * and experimental studies. ['-l o ] Since its first observation in 1991[111 La,@C,, has been widely known as a representative and abundant dimetallofullerene. Despite several attempts, its cage structure and symmetry have not yet been experimentally confirmed. In addition, it is currently of increasing interest whether encapsulated metal atoms are rigidly attached to fullerene cages or move about freely. Although rotation of a molecule within a cage is of great help in designing functional molecular devices,['21 very little has been known about the dynamic behavior of metal atoms encapsulated inside carbon cages. Based on 13C and 13'La NMR spectra of La,@CEO, we now report its structural determination and the first experimental evidence for circular motion of two La atoms.For the C,, fullerene there are seven distinct isomers (D,, D,,, Czv, CZv., D,, D,,, and Zh) that satisfy the isolated-pentagon rule.[131 The isomer distribution has been an open question, since C,, was a "missing" fullerene between C,, and C,, . By isolating C,, and analyzing the observed I3C NMR lines, however, it was verified that the D, isomer is most abundantly produced ( > 90 %) .[I4] It seems plausible to assume that two La atoms are encapsulated inside the D, isomer. However, theoretical calculationsl8I have shown that encapsulation of two La atoms inside the most unstable Z, cage (about 52 kcal mol-' less stable than the D, isomer) is most favorable (63 kcalmol-' more stable than encapsulation inside the D, isomer). This is because the I,,-symmetrical C,, has only two electrons in the fourfold degenerate HOMO and can accommodate six more electrons to form the stable, closed-shell electronic state of (La3'),C:;with a large HOMO-LUMO gap. The most stable endohedral structure optimized with the I, cage is shown in Figure 1. It has D,, symmetry; two La atoms are located equivalently on the C, axis facing the hexagonal rings of C,, with a long La-La distance of 3.655 A.r81 The 139La NMR spectrum of La,@C...

The backbone 1H, 13C, and 15N resonances of the c-Ha-Ras protein [a truncated version consisting of residues 1-171, Ras(1-171)] bound with GMPPNP (a slowly hydrolyzable analogue of GTP) were assigned and compared with those of the GDP-bound Ras(1-171). The backbone amide resonances of amino acid residues 10-13, 21, 31-39, 57-64, and 71 of Ras(1-171).GMPPNP, but not those of Ras(1-171).GDP, were extremely broadened, whereas other residues of Ras(1-171).GMPPNP exhibited amide resonances nearly as sharp as those of Ras(1-171). GDP. The residues exhibiting the extreme broadening, except for residues 21 and 71, are localized in three functional loop regions [loops L1, L2 (switch I), and L4 (switch II)], which are involved in hydrolysis of GTP and interactions with other proteins. From the temperature and magnetic field strength dependencies of the backbone amide resonance intensities, the extreme broadening was ascribed to the exchange at an intermediate rate on the NMR time scale. It was shown that the Ras(1-171) protein bound with GTP or GTPgammaS (another slowly hydrolyzable analogue of GTP) exhibits the same type of broadening. Therefore, it is a characteristic feature of the GTP-bound form of Ras that the L1, L2, and L4 loop regions, but not other regions, are in a rather slow interconversion between two or more stable conformers. This phenomenon, termed a "regional polysterism", of these loop regions may be related with their multifunctionality: the GTP-dependent interactions with several downstream target groups such as the Raf and RalGDS families and also with the GTPase activating protein (GAP) family. In fact, the binding of Ras(1-171).GMPPNP with the Ras-binding domain (residues 51-131) of c-Raf-1 was shown to eliminate the regional polysterism nearly completely. It was indicated, therefore, that each target/regulator selects its appropriate conformer among those presented by the "polysteric" binding interface of Ras. As the downstream target groups exhibit no apparent sequence homology to each other, it is possible that one target group prefers a conformer different from that preferred by another group. The involvement of loop L1 in the regional polysterism might suggest that the negative regulators, GAPs, bind to the polysteric binding interface (loops L2 and L4) of Ras and cooperatively select a conformer suitable for transition of the GTPase catalytic center, involving loops L1 and L4, into the highly active state.