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.

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform (13)C and (15)N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the beta-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D (1)H-(15)N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D (15)N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.

Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions,itisindispensable to observe their threedimensional (3D) structures at the atomic level in aphysiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/ baculovirus system using NMR data from living cells exclusively.The method was applied to five proteins,rat calmodulin, human HRas,h uman ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein GB 1d omain. In all cases,w ec ould obtain structural information from wellresolved in-cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be astandard tool for protein structure determinations in living eukaryotic cells.F or three proteins,w ea chieved well-converged 3D structures.A mong these,t he in-cell structure of protein GB 1 domain was most accurately determined, demonstrating that ah elix-loop region is tilted away from a b-sheet compared to the conformation in diluted solution.Biomacromolecules occupy as ignificant fraction of the intracellular volume (resulting in molecular crowding) [1] in which proteins are exposed to the excluded-volume effect, specific and non-specific interactions,a nd various dynamic intracellular processes. [2] Their biophysical properties under these effects,p articularly their molecular structures at the atomic level, are not fully understood. Therefore,i ti s indispensable to elucidate their native structures and dynamics in the physiologically natural environment inside cells,and to determine whether there are differences in the threedimensional (3D) structures of the biomacromolecules in cells compared to their diluted solution state.I n-cell NMR [3] is currently the only tool with which to observe proteins and deoxyribonucleic acid (DNA) at atomic resolution in living biological systems.I ta lso provides direct observations of protein behaviors in conjunction with chemical compounds that are potential targets for drug screening inside cells. [2a,4] Although in-cell NMR studies in various eukaryotic cells have become possible by either expressing target proteins inside cells [4] or by introducing stable isotope-enriched proteins from outside, [2a, 5] high-resolution protein 3D structures have been determined only in Escherichia coli cells. [6] To date,t he achievable target-protein concentration in eukaryotic cells was too low to obtain as ufficient number of nuclear Overhauser effect (NOE)-derived distance restraints.I nt he meantime,in-cell NMR studies of human-cultured cells have revealed that the intracellular environment does indeed influence the protein folding stability [5f] and reduces the volume occupied by intrinsically disordered proteins. [7] Recently,p rotein global folds in cells were obtained by exploiting NMR chemical shifts and paramagnetic NMR effects induced by intracellularly stable lanthanoid-binding t...

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional a-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family. p53, a well-known tumor suppressor that is mutated in more than 50% of all human tumors, induces genes leading either to cell-cycle arrest or to apoptosis. The discovery of two proteins with a high sequence identity to p53, called p63 and p73, has sparked speculations that tumor suppression is carried out by the combined action of several members of this protein family, for example, by direct interaction through heterooligomerization. Knockout mouse studies with p63 and p73, the two ancestral members, 1 have shown functional roles distinct from p53: p63 is essential for maintaining epithelial stem cells 2,3 and for protecting the genomic stability of oocytes, 4 whereas p73 is involved in neurogenesis, sensory pathways and homeostatic control. 5 p73 is further known as an important inducer of apoptosis in response to DNA damage. 6 Although only few mutations of p63 and p73 have been found in human tumors so far, overexpression of p63 is often observed in squamous cell carcinoma, 7-10 which has been shown to suppress p73-dependent apoptosis. 11 Among all p53 family members, including those from invertebrate species, the DNA binding domain is the most conserved domain, 12-15 followed by the oligomerization domain (OD), which is indispensable for the biological function of all p53 protein family members. [16][17][18] It is a structural domain that forms a tetramer, and mutations within the OD that inhibit tetramerization of p53 result in greatly reduced transcriptional activity. 19 In addition, several protein-protein interactions and posttranslational modifications require the tetrameric state as well, 16 and mutations in the OD of p53 that prevent oligomerization have been identified in human cancers. 20,21 Owing to its functional importance, the OD of p53 has been the target of several structure determination projects. 22,23 The p53 tetramer consists of a dimer of dimers, with each monomer contributing one b-strand and o...

The γ-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid β peptide whose aggregates form the senile plaques encountered in the brains of patients. Γ-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of γ-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.cell-free protein expression | gamma secretase | intramembrane proteolysis | membrane protein structure A lzheimer's disease is the most common form of dementia and affects more than 25 million people worldwide. The most characteristic histological feature of Alzheimer's disease is the presence of long, insoluble amyloid fibrils composed of amyloid β (Aβ) peptide which, either alone or as reservoirs for soluble Aβ oligomers (1, 2), appear to be the primary species responsible for the massive neuronal injury presented in patients. Aβ generation is categorized under an unusual physiological phenomenon termed regulated intramembrane proteolysis. Here, the amyloid precursor protein first sheds its ectodomain mediated by β-secretase. The remaining membrane-bound C-terminal fragment is subsequently processed at a γ-cleavage site by the γ-secretase complex, a multisubunit protease whose minimal essential components include presenilin 1 (PS1) or presenilin-2 (PS2), anterior pharynx-defective, nicastrin, and presenilin enhancer 2 (3). The pathological relevance of this final step lies in the observation that γ-cleavage is variable and can occur after three distinct positions, 38, 40, and 42, whose selection influences the self-aggregating potential of the secreted Aβ peptide. Aβ42, although the minor species, appears to show the strongest potency for oligomerization and represents the majority of Aβ in amyloid plaques (4). Over 150 familial Alzheimer's disease associated mutations (www.molgen.ua.ac.be/ADMutations) have been linked to PS1, the catalyt...

Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells.

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.