Showing papers by "Christian Griesinger published in 2014"

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

Abstract: The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

The oligomer modulator anle138b inhibits disease progression in a Parkinson mouse model even with treatment started after disease onset

Abstract: ]. parkinson’s disease (pD) is characterized by the deposition of aggregated alpha-synuclein (asyn). recent evidence suggests that oligomers formed in the aggregation process constitute the main toxic species causing neurodegeneration. recently, we reported that the oligomer modulator “anle138b” [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole] is capable of prolonging the survival of prion-infected mice and of various animal models of pD [5blocked formation and accumulation of asyn oligomers in the brain, reduced disease-associated motor deficits, and led to prolonged disease-free survival [5 ]. These findings support the following

Long-range correlated dynamics in intrinsically disordered proteins.

Abstract: Intrinsically disordered proteins (IDPs) are involved in a wide variety of physiological and pathological processes and are best described by ensembles of rapidly interconverting conformers. Using fast field cycling relaxation measurements we here show that the IDP α-synuclein as well as a variety of other IDPs undergoes slow reorientations at time scales comparable to folded proteins. The slow motions are not perturbed by mutations in α-synuclein, which are related to genetic forms of Parkinson’s disease, and do not depend on secondary and tertiary structural propensities. Ensemble-based hydrodynamic calculations suggest that the time scale of the underlying correlated motion is largely determined by hydrodynamic coupling between locally rigid segments. Our study indicates that long-range correlated dynamics are an intrinsic property of IDPs and offers a general physical mechanism of correlated motions in highly flexible biomolecular systems.

Site-Specific Copper-Catalyzed Oxidation of α-Synuclein: Tightening the Link between Metal Binding and Protein Oxidative Damage in Parkinson’s Disease

Abstract: Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinson’s disease (PD). CuII binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific CuI-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, CuI binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif 1MDVFM5 constitute key structural determinants for the high-affinity binding of CuI to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for CuI, whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-CuI complex, whereas Met-116 and Met-127 in the C-terminal region r...

Graphene oxide liquid crystals as a versatile and tunable alignment medium for the measurement of residual dipolar couplings in organic solvents.

Abstract: Residual dipolar couplings (RDCs) have proven to be an invaluable anisotropic NMR parameter for the structural elucidation of complex biopolymers and organic molecules. However, a remaining bottleneck limiting its wider use by organic and natural product chemists is the lack of a range of easily applicable aligning media for diverse organic solvents. In this study, graphene oxide (GO) liquid crystals (LCs) were developed to induce partial orientation of organic molecules to allow RDC measurements. These LCs were determined to be maintainable at very low concentrations (as low as 1 mg/mL, corresponding to quadrupolar 2H splittings ranging from 2.8 to 30 Hz and maximum 13C–1H dipolar couplings of 20 Hz for camphor in a CH3COCH3/water system) and to be remarkably stable and broadly compatible with aqueous and organic solvents such as dimethyl sulfoxide, CH3COCH3, and CH3CN. Moreover, compared with those for other alignment media, very clean and high-quality NMR spectra were acquired with the GO molecules in ...

A designed conformational shift to control protein binding specificity.

Abstract: In a conformational selection scenario, manipulating the populations of binding-competent states should be expected to affect protein binding. We demonstrate how in silico designed point mutations within the core of ubiquitin, remote from the binding interface, change the binding specificity by shifting the conformational equilibrium of the ground-state ensemble between open and closed substates that have a similar population in the wild-type protein. Binding affinities determined by NMR titration experiments agree with the predictions, thereby showing that, indeed, a shift in the conformational equilibrium enables us to alter ubiquitin’s binding specificity and hence its function. Thus, we present a novel route towards designing specific binding by a conformational shift through exploiting the fact that conformational selection depends on the concentration of binding-competent substates.

relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data

Abstract: Nuclear Magnetic Resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy to use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the GUI, is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimisation time are granted by parallelisation for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another – using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis. Availability: The software relax is written in Python with C modules and is released under the GPLv3+ licence. Source code and precompiled binaries for all major operating systems are available from http://www.nmr-relax.com.

ORIUM: optimized RDC-based Iterative and Unified Model-free analysis.

Abstract: Residual dipolar couplings (RDCs) are NMR parameters that provide both structural and dynamic information concerning inter-nuclear vectors, such as N–HN and Cα–Hα bonds within the protein backbone Two approaches for extracting this information from RDCs are the model free analysis (MFA) (Meiler et al in J Am Chem Soc 123:6098–6107, 2001; Peti et al in J Am Chem Soc 124:5822–5833, 2002) and the direct interpretation of dipolar couplings (DIDCs) (Tolman in J Am Chem Soc 124:12020–12030, 2002) Both methods have been incorporated into iterative schemes, namely the self-consistent RDC based MFA (SCRM) (Lakomek et al in J Biomol NMR 41:139–155, 2008) and iterative DIDC (Yao et al in J Phys Chem B 112:6045–6056, 2008), with the goal of removing the influence of structural noise in the MFA and DIDC formulations Here, we report a new iterative procedure entitled Optimized RDC-based Iterative and Unified Model-free analysis (ORIUM) ORIUM unifies theoretical concepts developed in the MFA, SCRM, and DIDC methods to construct a computationally less demanding approach to determine these structural and dynamic parameters In all schemes, dynamic averaging reduces the actual magnitude of the alignment tensors complicating the determination of the absolute values for the generalized order parameters To readdress this scaling issue that has been previously investigated (Lakomek et al in J Biomol NMR 41:139–155, 2008; Salmon et al in Angew Chem Int Edit 48:4154–4157, 2009), a new method is presented using only RDC data to establish a lower bound on protein motion, bypassing the requirement of Lipari–Szabo order parameters ORIUM and the new scaling procedure are applied to the proteins ubiquitin and the third immunoglobulin domain of protein G (GB3) Our results indicate good agreement with the SCRM and iterative DIDC approaches and signify the general applicability of ORIUM and the proposed scaling for the extraction of inter-nuclear vector structural and dynamic content

De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy

Abstract: We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans DXS as a model enzyme, targeting the thiamine diphosphate (TDP) pocket of DXS whilst also exploring the putative substrate-binding pocket, where selectivity over other human TDP-dependent enzymes could be gained. The STI methodology – suitable for weak binders – was essential to determine the binding mode in solution of one of the fragments, circumventing the requirement for an X-ray co-crystal structure, which is known to be particularly challenging for this specific enzyme and in general for weak binders. Based on this finding, we carried out fragment growing and optimisation, which led to a three-fold more potent fragment, about as potent as the well-established thiamine analogue deazathiamine. The STI methodology proved therefore its strong potential as a tool to support medicinal-chemistry projects in their early stages, especially when dealing with weak binders.

Macromolecular assembly of the adaptor SLP-65 at intracellular vesicles in resting B cells

Abstract: The traditional view of how intracellular effector proteins are recruited to the B cell antigen receptor (BCR) complex at the plasma membrane is based on the occurrence of direct protein-protein interactions, as exemplified by the recruitment of the tyrosine kinase Syk (spleen tyrosine kinase) to phosphorylated motifs in BCR signaling subunits By contrast, the subcellular targeting of the cytosolic adaptor protein SLP-65 (Src homology 2 domain–containing leukocyte adaptor protein of 65 kD), which serves as a proximal Syk substrate, is unclear We showed that SLP-65 activation required its association at vesicular compartments in resting B cells A module of ∼50 amino acid residues located at the amino terminus of SLP-65 anchored SLP-65 to the vesicles Nuclear magnetic resonance spectroscopy showed that the SLP-65 amino terminus was structurally disordered in solution, but could bind in a structured manner to noncharged lipid components of cellular membranes Our finding that preformed vesicular signaling scaffolds are required for B cell activation indicates that vesicles may deliver preassembled signaling cargo to sites of BCR activation