The oncoprotein E7 of human papilloma viruses (HPV) is involved in the pathogenesis and maintenance of human cervical cancers. The most prevalent HPV types found in cervix carcinomas are HPV16, 18 and 45. The structure of the E7 dimer from HPV45 (PDB 2F8B) was determined by nuclear magnetic resonance spectroscopy. Each monomer comprises an unfolded N-terminus and a well-structured C-terminal domain with a b1b2a1b3a2 topology representing a unique zinc-binding fold found only for E7. Dimerization occurs through the a1/a1 0 helices and intermolecular b-sheet formation but excludes the zinc-binding sites. E7 is reported to interact with a number of cellular proteins (e.g. pRb, p21 CIP1 ). Binding of a peptide derived from the C-terminus of p21CIP1 to the Cterminal domain of E7 was characterized by monitoring chemical shift perturbations of the amide groups of E7. This provides direct evidence that a shallow groove situated between a1 and b1 of the E7 C-terminal domain is interacting with the C-terminus of p21 CIP1. Intriguingly, this binding site overlaps with the low-affinity binding site on E7 for the C-domain of pRb.

Knowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize— and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies.

Fast (>100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Here we apply this technique to RNA and assign nucleotide spin systems through highly sensitive multidimensional experiments.

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

Intranucleotide resonance of the 26mer box C/D RNA in complex with the L7Ae protein were assigned by solid-state NMR (ssNMR; see picture) spectroscopy. This investigation opens the way for studying RNA in large protein-RNA complexes by ssNMR spectroscopy.

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.

Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.

Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα.