Martina Huber

In vivo visualization of gene expression using magnetic resonance imaging

High-resolution in vivo imaging of gene expression is not possible in opaque animals by existing techniques. Here we present a new approach for obtaining such images by magnetic resonance imaging (MRI) using an MRI contrast agent that can indicate reporter gene expression in living animals. We have prepared MRI contrast agents in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal. Here, we report an agent where galactopyranose is the blocking group. This group renders the MRI contrast agent sensitive to expression of the commonly used marker gene, β-galactosidase. To cellular resolution, regions of higher intensity in the MR image correlate with regions expressing marker enzyme. These results offer the promise of in vivo mapping of gene expression in transgenic animals and validate a general approach for constructing a family of MRI contrast agents that respond to biological activity.

Antiparallel Arrangement of the Helices of Vesicle-Bound α-Synuclein

alpha-Synuclein (alphaS) is the main component of Lewy bodies from Parkinsons disease. That alphaS binds to membranes is known, but the conformation it adopts is still unclear. Pulsed EPR on doubly spin-labeled variants of alphaS sheds light on the most likely structure. For alphaS bound to vesicles large enough to accommodate also the extended conformation, an antiparallel helix conformation is found. This suggests that the bent structure shown is the preferred conformation of alphaS on membranes.

A pulsed EPR method to determine distances between paramagnetic centers with strong spectral anisotropy and radicals: the dead-time free RIDME sequence.

Methods to determine distances between paramagnetic metal centers and radicals are scarce. This is unfortunate because paramagnetic metal centers are frequent in biological systems and so far have not been employed much as distance markers. Successful pulse sequences that directly target the dipolar interactions cannot be applied to paramagnetic metal centers with fast relaxation rates and large g-anisotropy, if no echos can be detected and the excitation bandwidth is not sufficient to cover a sufficiently large part of the spectrum. The RIDME method Kulik et al. (2002) [20] circumvents this problem by making use of the T(1)-induced spin-flip of the transition-metal ion. Designed to measure distance between such a fast relaxing metal center and a radical, it suffers from a dead time problem. We show that this is severe because the anisotropy of the metal center broadens the dipolar curves, which therefore, only can be analyzed if the full curve is known. Here, we introduce five-pulse RIDME (5p-RIDME) that is intrinsically dead-time free. Proper functioning of the sequence is demonstrated on a nitroxide biradical. The distance between a low-spin Fe(III) center and a spin label in spin-labeled cytochrome f shows the complete dipolar trace of a transition-metal ion center and a spin label, yielding the distance expected from the structure.

Spin-Label EPR on α-Synuclein Reveals Differences in the Membrane Binding Affinity of the Two Antiparallel Helices

The putative function of the Parkinsons disease‐related protein α‐Synuclein (αS) is thought to involve membrane binding. Therefore, the interaction of αS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of αS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but αS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative‐charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of αS. Thus, the interaction of αS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of αS to membranes. These findings contribute to our understanding of the details and structural consequences of αS–membrane interactions.

Direct Evidence of Coexisting Horseshoe and Extended Helix Conformations of Membrane-Bound Alpha-Synuclein

IDPs lack a well-defined three-dimensional fold anddisplay remarkable conformational flexibility. This property po-tentially enables them to be promiscuous in their interactionsand to adapt their structure according to the needed function.In the case of aS, the protein is capable of adopting a b-sheetstructure in the amyloid fibrils constituting the Lewy bodiesand an a-helical structure in the membrane bound form. Theexact physiological role of aS has yet to be determined, butmembrane binding seems to be important for its function.

Electronic structure of the oxidized primary electron donor of the HL (M202) and HL (L173) heterodimer mutants of the photosynthetic bacterium Rhodobacter sphaeroides: ENDOR on single crystals of reaction centers

Abstract The electronic structure of the primary electron donor (D) in the heterodimer mutants mutants HL (M202) and HL (L173) of the photosynthetic bacterium Rhodobacter sphaeroides was investigated using EPR and ENDOR (electron nuclear double resonance) methods on single crystals of reaction centers. In the mutants, one of the two bacteriochlorophyll (BChl) molecules of D is replaced by a bacteriopheophytin. The assignment of the ENDOR lines to specific methyl and non-methyl protons was accomplished by comparing that directions of the principal axes of the hyperfine tensors with the directions predicted from the X-ray structure and theory. We showed that the unpaired electron is localized on the BChl in the heterodimers, i.e., on the L-side (D L ) in the HL (M202) and on the M-side (D M ) in the HL (L173) mutant. Significant differences in the electronic structure of D L and D M were observed; they are attributed to the protein and/or pigment environment. Possible consequences of these differences for electron transfer, e.g., unidirectionality are discussed. The inequivalence of D L and D M also shows up in the asymmetry of the electronic structure of D in the native homodimer, whose electronic structure was reinterpreted using the heterodimers as monomer models.

A Stable Lipid-Induced Aggregate of alpha-Synuclein

The Parkinsons disease-related protein alpha-Synuclein (alphaS) is a 140 residue intrinsically disordered protein. Its membrane-binding properties are thought to be relevant for its physiological or pathologic activity. Here, the interaction of alphaS with POPG [1-Palmitoyl-2-Oleoyl-sn-Glycero-3-(Phosphorac-(1-glycerol))] small unilamellar vesicles (SUVs) is investigated by spin-label EPR using double electron-electron resonance (DEER). Intermolecular distances between four single mutants reveal that well-defined aggregates are formed. The data suggest a coexistence of two dimer structures with main interactions in the helix 2, encompassing residues 50-100. Previously, the horseshoe conformation was detected by intramolecular restraints obtained by DEER on alphaS double mutants (Drescher et al. J. Am. Chem. Soc. 2008, 130, 7796). The present study suggests that interdigitation of two monomers in the aggregate fills the void between the two helices of each of the monomers thus providing a rationale for the horseshoe structure. This aggregate is lipid induced and affects the structure of the POPG SUVs, which become leaky and diminish in size upon contact with alphaS suggesting a possible origin of conflicting results in the recent literature (Jao et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (50), 19666; Georgieva et al. J. Am. Chem. Soc. 2008, 130 (39), 12856; Bortolus et al. J. Am. Chem. Soc. 2008, 130, 6690).

Hunting the Chameleon : Structural Conformations of the Intrinsically Disordered Protein Alpha-Synuclein

The human a-synuclein protein (ASYN) plays a central role in the etiology of Parkinson’s disease, and forms fibrillar aggregates that are found in Lewy bodies and Lewy neurites in the brain, structures which are the hallmark of the disease. Three point mutations (A30P, A53T, and E46K) are associated with early-onset Parkinson’s disease. Modifications such as phosphorylation of the serine residue at position 129, and truncations of the protein, are reported to play an important role in the toxicity of ASYN. ASYN displays remarkable structural versatility: it has long been considered an intrinsically disordered or “natively unfolded” protein at physiological conditions, but can readily adopt b-sheet structure in aggregates or a-helical structure when bound to lipids. Very recent reports suggest that at physiological conditions in vivo, ASYN adopts a helical tetrameric structure, although this observation remains controversial. The intriguing reports about the ASYN tetramer could imply that these structures are yet another face of the chameleon-like nature of the protein in vivo. The notion of a-synuclein as a protein chameleon was introduced in 2003 by Uversky, and the ensuing years have only served to reinforce this view of the protein. The function of ASYN is unknown, but it is thought to involve lipid-binding in vesicles and synaptic membranes. The intrinsically disordered nature of the protein renders it intractable to standard high-resolution structural biology methods, making single-molecule Fçrster resonance energy transfer (FRET) and spectroscopic approaches that yield distance constraints invaluable in establishing structural parameters for the protein. In general for amyloid diseases, early aggregation intermediates are suspected of playing a key role in cell damage, although the structures and mechanisms of action of these early intermediates are unknown. There is evidence that amyloid toxicity may be caused by membrane permeabilization by pore-like early intermediates leading to disruption of calcium homeostasis and cell-death; such pores have been detected for ASYN, 45–48] but the hypothesis remains to be unequivocally verified. Thus, it is very likely that the structural diversity of ASYN is a key element in the pathology of the synucleinopathies.

Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.

Endor and triple resonance in solutions of the chlorophyll a and bis(chlorophyll)cyclophane radical cations

Abstract The radical cations of chlorophyll a (Chl a) and bis ( chlorophyll )cyclophane (BCP), a synthetic model dimer of chlorophyll, were studied by ENDOR and TRIPLE resonance in liquid solution. The measured hyperiine coupling constants (hfcs) of Chl a + were compared with the results of molecular orbital calculations. By comparing the hfcs of BCP + and Chl a + a localization of the unpaired electron on one macrocycle of BCP + is deduced.