Jochen Junker

The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli.

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.

[2] – Cross-Correlated Relaxation for Measurement of Angles between Tensorial Interactions

c Theory, experimental aspects, and use in structure calculation of cross-correlated relaxation rates measured on zeroand doublequantum coherences in liquid state NMR are presented. The relative size of the interaction depends on the projection angle between the two tensorial interactions. The tensorial interaction can be either a dipolar interaction or a chemical shift anisotropy relaxation mechanism (CSA). Effects of additional sources of relaxation on the cross-correlated relaxation rates are analyzed. Also, an easy-to-use formalism is given to manipulate different cross-correlated relaxation interactions. The application addresses measurement of the backbone angle c in a protein by measuring dipole(N–H)–dipole(C–H) and CSA(N)–dipole(C– H) cross-correlated relaxation rates. It is shown that ambiguities due to the 3 cosu-1 dependence of one cross-correlated relaxation rate can be overcome by measuring additional cross-correlated relaxation rates. The use of cross-correlated relaxation rates is demonstrated in structure calculations.

Structure Determination of a Key Intermediate of the Enantioselective Pd Complex Catalyzed Allylic Substitution Reaction

The structure of a catalytic intermediate with important implications for the interpretation of the stereochemical outcome of the palladium complex catalyzed allylic substitution with phosphino-oxazoline (PHOX) ligands is determined by liquid state NMR. The complex displays a novel structure that is highly distorted compared with other palladium eta2-olefin complexes known so far. The structure has been determined from nuclear overhauser data (NOE), scalar coupling constants, and long range projection angle restraints derived from dipole dipole cross-correlated relaxation of multiple quantum coherence. The latter restraints have been implemented into a distance geometry protocol. The projection angle restraints yield a higher precision in the determination of the relative orientation of the two molecular moieties and are essential to provide an exact structural definition of the olefinic part of the catalytic intermediate with respect to the ligand.

TAR-RNA recognition by a novel cyclic aminoglycoside analogue

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.

Validation of structural proposals by substructure analysis and 13C NMR chemical shift prediction.

The 2D NMR-guided computer program COCON can be extremely valuable for the constitutional analysis of unknown compounds, if its results are evaluated by neural network-assisted 13C NMR chemical shift and substructure analyses. As instructive examples, data sets of four differently complex marine natural products were thoroughly investigated. As a significant step towards a true automated structure elucidation, it is shown that the primary COCON output can be safely diminished to less than 1% of its original size without losing the correct structural proposal.

Tools and databases to analyze protein flexibility; Approaches to mapping implied features onto sequences

Publisher Summary This chapter describes the way protein flexibility can be analyzed statistically in a database. The database of macromolecular movements, which is accessible over the Internet, organizes a few hundred well-characterized motions on the basis of size and then packing, with the involvement of a well-packed interface in the motion being a key classifying feature. The chapter describes the computational tools employed in the database analysis—namely, (1) structure comparison, which is useful to align and superpose different conformations, (2) adiabatic mapping interpolation, which is implemented on a large scale by the morph server, provides movie-like pathways between two superposed conformations, and in the process, generates many standardized statistics, (3) normal mode analysis, which provides readily interpretable information about the flexibility of a single conformation, and (4) Voronoi volume calculations, which provide a rigorous basis for characterizing packing. The chapter also explains the way structural features in the motions database can be related to sequence, an important part of the overall process of transferring annotation to uncharacterized genomic data. This allows determination of a sequence-propensity scale for amino acids to be in linkers in general or flexible hinges in particular.

Global perspectives on proteins: comparing genomes in terms of folds, pathways and beyond

The sequencing of complete genomes provides us with a global view of all the proteins in an organism. Proteomic analysis can be done on a purely sequence-based level, with a focus on finding homologues and grouping them into families and clusters of orthologs. However, incorporating protein structure into this analysis provides valuable simplification; it allows one to collect together very distantly related sequences, thus condensing the proteome into a minimal number of ‘parts.’ We describe issues related to surveying proteomes in terms of structural parts, including methods for fold assignment and formats for comparisons (eg top-10 lists and whole-genome trees), and show how biases in the databases and in sampling can affect these surveys. We illustrate our main points through a case study on the unique protein properties evident in many thermophile genomes (eg more salt bridges). Finally, we discuss metabolic pathways as an even greater simplification of genomes. In comparison to folds these allow the organization of many more genes into coherent systems, yet can nevertheless be understood in many of the same terms.

Theoretical NMR correlations based Structure Discussion

The constitutional assignment of natural products by NMR spectroscopy is usually based on 2D NMR experiments like COSY, HSQC, and HMBC. The actual difficulty of the structure elucidation problem depends more on the type of the investigated molecule than on its size. The moment HMBC data is involved in the process or a large number of heteroatoms is present, a possibility of multiple solutions fitting the same data set exists. A structure elucidation software can be used to find such alternative constitutional assignments and help in the discussion in order to find the correct solution. But this is rarely done. This article describes the use of theoretical NMR correlation data in the structure elucidation process with WEBCOCON, not for the initial constitutional assignments, but to define how well a suggested molecule could have been described by NMR correlation data. The results of this analysis can be used to decide on further steps needed to assure the correctness of the structural assignment. As first step the analysis of the deviation of carbon chemical shifts is performed, comparing chemical shifts predicted for each possible solution with the experimental data. The application of this technique to three well known compounds is shown. Using NMR correlation data alone for the description of the constitutions is not always enough, even when including 13C chemical shift prediction.