Frank D. Sönnichsen

Magnetic Bistability of Molecules in Homogeneous Solution at Room Temperature

A photoresponsive ligand is used to reversibly modulate the magnetic properties of a nickel compound. Magnetic bistability, as manifested in the magnetization of ferromagnetic materials or spin crossover in transition metal complexes, has essentially been restricted to either bulk materials or to very low temperatures. We now present a molecular spin switch that is bistable at room temperature in homogeneous solution. Irradiation of a carefully designed nickel complex with blue-green light (500 nanometers) induces coordination of a tethered pyridine ligand and concomitant electronic rearrangement from a diamagnetic to a paramagnetic state in up to 75% of the ensemble. The process is fully reversible on irradiation with violet-blue light (435 nanometers). No fatigue or degradation is observed after several thousand cycles at room temperature under air. Preliminary data show promise for applications in magnetic resonance imaging.

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and α-helical prion protein, PrPC, to the β-sheet-rich PrPSc. This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrPC monomer, structures of the pathogenic PrPSc or synthetic PrPSc-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrPC. Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrPC involves major refolding of the C-terminal α-helical region. The core of the amyloid maps to C-terminal residues from ≈160–220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of β-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to β-structure.

Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase

Opening the Portico Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane phosphotransferases that function in prokaryotic-specific metabolic pathways. Van Horn et al. (p. 1726) determined the structure of the 40-kilodalton functional homotrimer of E. coli DAGK by solution nuclear magnetic resonance spectroscopy. Each monomer comprises three transmembrane helices. The third transmembrane helix from each subunit is domain-swapped to pack against the first and second transmembrane helices from an adjacent subunit. These three helices frame a portico-like membrane-submerged cavity that contains residues critical for activity in close proximity to residues critical for folding. The structure provides insight into the determinants of lipid substrate specificity and phosphotransferase activity. Mutations reveal the distribution of sequence changes that alter folding and affect function in a membrane-bound enzyme. Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.

Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.

BACKGROUND Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

Solution structure of the E200K variant of human prion protein. Implications for the mechanism of pathogenesis in familial prion diseases.

Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrPC, into a pathogenic conformer, PrPSc. Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrPC → PrPSc conversion in the E200K form of hereditary prion disease.

On choosing a detergent for solution NMR studies of membrane proteins

Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (× 10-7cm2/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggesting that this well-known protein denaturant should be used with care in studies of complex membrane proteins.

Reversed-phase chromatography of synthetic amphipathic α-helical peptides as a model for ligand/receptor interactions Effect of changing hydrophobic environment on the relative hydrophilicity/hydrophobicity of amino acid side-chains

To mimic a hydrophobic protein binding domain, which is a region on the surface of a protein that has a preference or a specificity to interact with a complementary surface, we have designed amphipathic alpha-helical peptides where the non-polar face interacts with the non-polar surface of a reversed-phase stationary phase. Two series of potentially amphipathic alpha-helical peptides, a native Ala peptide (AA9) and a native Leu peptide (LL9), were designed where the native peptide contains 7 residues of either Ala or Leu, respectively, in its non-polar face. This design results in an overall hydrophobicity of the non-polar face of the Leu peptide that is greater than that of the non-polar face of the native Ala peptide. Mutants of the native Ala-face peptide, AX9, and the native Leu-face peptide, LX9, were designed by replacing one residue in the centre of the non-polar face in both series of peptides. Therefore, by changing the hydrophobicity of the environment surrounding the mutated amino acid side-chain, the effect on the hydrophilicity/hydrophobicity of each amino acid side-chain could be determined. Using the substitutions Ala, Leu, Lys and Glu, it was shown that the maximum hydrophilicity of these amino acid side-chains could be determined when the environment surrounding the mutation is maximally hydrophobic; whereas its maximum hydrophobicity can be determined when the environment surrounding the mutation is minimally hydrophobic. This procedure was further extended to the remaining amino acids commonly found in proteins and it was determined that this general principle applies to all 20 amino acids. These results have major implications to understanding the hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.

Mass and NMR spectroscopic characterization of 3,4-methylenedioxypyrovalerone: A designer drug with α-pyrrolidinophenone structure

This study presents and discusses the nuclear magnetic resonance (NMR) spectroscopic and mass spectroscopic data of the designer drug 3,4-methylenedioxypyrovalerone (MDPV), a drug variant of pyrovalerone. MDPV was first seized in Germany in the year 2007. The structure elucidation of the aliphatic part of MDPV was carried out by product ion spectroscopy of the immonium ion with m/z 126 formed after electron ionization, and by 1D (1)H and (13)C NMR spectroscopy. Additional two-dimensional NMR spectroscopy was used to verify the structure of the alkyl side chain, and to determine the methylenedioxy position in the aromatic ring.

Coordination‐Induced Spin Crossover (CISCO) through Axial Bonding of Substituted Pyridines to Nickel–Porphyrins: σ‐Donor versus π‐Acceptor Effects

Nickel-porphyrins, with their rigid quadratic planar coordination framework, provide an excellent model to study the coordination-induced spin crossover (CISCO) effect because bonding of one or two axial ligands to the metal center leads to a spin transition from S=0 to S=1. Herein, both equilibrium constants K(1S) and K(2), and for the first time also the corresponding thermodynamic parameters DeltaH(1S), DeltaH(2), DeltaS(1S), and DeltaS(2), are determined for the reaction of a nickel-porphyrin (Ni-tetrakis(pentafluorophenyl)porphyrin) with different 4-substituted pyridines by temperature-dependent NMR spectroscopy. The association constants K(1S) and K(2) are correlated with the basicity of the 4-substituted pyridines (R: OMe>H>CO(2)Et>NO(2)) whereas the DeltaH(1S) values exhibit a completely different order (OMeCO(2)Et>NO(2)). 4-Nitropyridine exhibits the largest binding enthalpy, which, however, is overcompensated by a large negative binding entropy. We attribute the large association enthalpy of nitropyridine with porphyrin to the back donation of electrons from the Ni d(xz) and d(yz) orbitals into the pi orbitals of pyridine, and the negative association entropy to a decrease in vibrational and internal rotation entropy of the more rigid porphyrin-pyridine complex. Back donation for the nitro- and cyanopyridine complexes is also confirmed by IR spectroscopy, and shows a shift of the N-O and C-N vibrations, respectively, to lower wave numbers. X-ray structures of 2:1 complexes with nitro-, cyano-, and dimethylaminopyridine provide further indication of a back donation. A further trend has been observed: the more basic the pyridine the larger is K(1S) relative to K(2). For nitropyridine K(2) is 17 times larger than K(1S) and in the case of methoxypyridine K(2) and K(1S) are almost equal.

ESCHERICHIA COLI DIACYLGLYCEROL KINASE : A CASE STUDY IN THE APPLICATION OF SOLUTION NMR METHODS TO AN INTEGRAL MEMBRANE PROTEIN

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.