Showing papers by "Christian Griesinger published in 2005"

Release of long-range tertiary interactions potentiates aggregation of natively unstructured α-synuclein

Abstract: In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein α-synuclein (αS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, αS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric αS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of αS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

Structural characterization of copper(II) binding to α-synuclein: Insights into the bioinorganic chemistry of Parkinson's disease

Abstract: The aggregation of α-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen/oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS–Cu(II) complexes reveals the existence of conformational restrictions in the native state of the protein. The metallobiology of Cu(II) in Parkinson's disease is discussed by a comparative analysis with other Cu(II)-binding proteins involved in neurodegenerative disorders.

Defining Long-Range Order and Local Disorder in Native α-Synuclein Using Residual Dipolar Couplings

Abstract: Intrinsically unstructured proteins play key biochemical roles in a vast range of normal and pathological processes. To study these systems, it is necessary to invoke an ensemble of rapidly interconverting conformations. Residual dipolar couplings (RDCs) are particularly powerful probes of the behavior of unfolded proteins, reporting on time and ensemble-averaged conformations up to and beyond the millisecond time scale. In this study, we present a novel interpretation of RDCs in unfolded systems that simultaneously defines long-range structural order and local conformational sampling. This approach is used to describe the structure and dynamics of α-Synuclein (αS), a protein that is strongly implicated in the development of Parkinson's disease (PD), allowing unambiguous detection of strongly populated conformers containing long-range contacts between the N- and C-terminal domains. The structural model combines two features required for the description of αS in solution: local conformational fluctuation ...

The INPHARMA method: protein-mediated interligand NOEs for pharmacophore mapping.

Abstract: In structure-based drug design, the relative orientationof twocompetitive ligands A and B in the receptor binding pocketplays a central role in the design of high-affinity drugcandidates from weakly bound lead compounds. In thiscommunication we report on the observation of interligandspin-diffusion-mediated transferred-NOE data, which havethe potential to be used for the determination of the relativeorientation of two competitive ligands in the receptor bindingpocket. In many cases, the orientation of one ligand in thebinding pocket is known, for example, from crystallographyor fluorescence resonance energy transfer (FRET) data. Inthese cases it is desirable to develop a methodology thatallows the determination of a unique binding mode andpharmacophore model for a second competitive ligand. Weanticipate that the interligand transferred NOEs describedhere could provide the experimental basis for a methodologythatis applicabletoanycombinationofligandsweaklyboundto a common receptor.The conformation of a ligand in the binding pocket of amacromolecular receptor can be accessed by NMR spectros-copyinsolutionbytwoapproaches.Ifthemolecularweightofthe complex is of the order of 50 kDa or smaller and

Convenient Synthesis of Multifunctional EDTA-Based Chiral Metal Chelates Substituted with an S-Mesylcysteine

Abstract: We describe the synthetic route to ethylenediaminetetraacetic acid (EDTA) derivatives that can be attached to surface-exposed thiol functional groups of cysteine residues in proteins, via a methylthiosulfonate moiety that is connected in a stereochemically unique way to the C-1 carbon atom of EDTA. Such compounds can be used to align proteins in solution without the need to add liquid crystalline media, and are, therefore, of great interest for the NMR spectroscopic analysis of biomolecules. The binding constant for the paramagnetic tag to lanthanide ions was determined by measuring luminescence. For the Tb+3–ligand complex, a Kb value of 6.5×1017 M−1 was obtained. This value is in excellent agreement with literature values for the related EDTA compound. In addition, it could be shown that there is no significant reduction in the luminescence intensity upon addition of a 104 excess of Ca2+ ions, indicating that this paramagnetic tag is compatible with buffers containing high concentrations of divalent alkaline earth ions.

Studying Molecular 3D Structure and Dynamics by High-Resolution Solid-State NMR: Application to l-Tyrosine-Ethylester

Abstract: A unified approach to the study of 3D conformation and molecular dynamics using magic-angle-spinning solid-state NMR is demonstrated on a uniformly 13C-labeled sample of l-tyrosine-ethylester.

Side‐Chain Orientation and Hydrogen‐Bonding Imprint Supra‐τc Motion on the Protein Backbone of Ubiquitin

Abstract: Knowledge about protein dynamics is crucial for the understanding of protein function. NMR spectroscopy can characterize the amplitudes and rates of motions that are either faster than the rotational correlation time tc (sub-tc motion) with heteronuclear relaxation experiments or between approximately 50 ms and 10 ms (ms/ms motion) with relaxation dispersion. 3] The extent of motions occurring in folded proteins on the time scale between the rotational correlation time tc and the ms/ms range (supra-tc motion) has been a matter of debate. However, the functional relevance of such motions has recently been shown for the aggregation rate of natively unfolded proteins involved in neurodegenerative diseases. Very recently, motions on this supra-tc time scale have been observed in a 0.2 ms molecular dynamics simulation of ubiquitin. Residual dipolar couplings (rdcs) were recognized early on as an ideal tool to widen the time window of dynamics that can be characterized by NMR spectroscopy, since they are sensitive to motional averaging occurring over the suband supra-tc time scales (ps to ms). [4] We recently analyzed NH rdcs of ubiquitin measured in 31 different alignment conditions and derived the order parameters Srdc 1⁄4 4p 5 P2

An asymmetric ion channel derived from gramicidin A. Synthesis, function and NMR structure.

Abstract: The biological ion channel gramicidin A (gA) was modified by synthetic means to obtain the tail-to-tail linked asymmetric gA-derived dimer compound 3. Single-channel current measurements for 3 in planar lipid bilayers exhibit an Eisenman I ion selectivity for alkali cations. The structural asymmetry does not lead to an observable functional asymmetry. The structure of 3 in solution without and with Cs cations was investigated by 1H-NMR spectroscopy. In CDCl3/CD3OH (1 : 1, v/v), 3 forms a mixture of double-stranded β-helices. Upon addition of excess CsCl, the double-stranded species are converted completely into one new conformer: the right-handed single-stranded β-helix. A combination of DQF-COSY and TOCSY was used for the assignment of the 1H-NMR spectrum of the Cs–3 complex in CDCl3/CD3OH (1 : 1, v/v). A total of 69 backbone, 27 long-range, and 64 side-chain distance restraints were obtained from NOESY together with 25 φ and 14 χ1 torsion angles obtained from coupling constants. These data were used as input for structure calculation with dyana built in sybyl 6.8. A final set of 11 structures with an average rmsd for the backbone of 0.45 A was obtained (PDB: 1TKQ). The structure of the Cs–3 complex in solution is equivalent to the bioactive channel conformation in the membrane environment.

The structure of hormaomycin and one of its all-peptide aza-analogues in solution: syntheses and biological activities of new hormaomycin analogues.

Abstract: Four new aza-analogues of hormaomycin 1, a secondary metabolite with interesting biological activities produced by Streptomyces griseoflavus, were synthesized and subjected to preliminary tests of their antibiotic activity to provide new insights into the structure-activity relationship studies of this class of compounds. The solution structures of hormaomycin 1 and its aza-analogue 2 a were determined by NMR spectroscopy. The data exhibited a reasonably rigid conformation for both molecules, stabilized by stacking interactions between the aromatic moieties attached to the ring and the side chain. According to NMR-spectral data the aza-analogue epi-2 a has a rather different conformation and indeed shows no antibacterial activity whatsoever.