Biomedical subjects

Stephan G Zech

Publications and source records attributed to Stephan G Zech.

9 recordsLinked to original sources

PubMed2006

Determination of the hydration number of gadolinium(III) complexes by high-field pulsed 17O ENDOR spectroscopy.

Pulsed 17O Mims electron-nuclear double resonance (ENDOR) spectroscopy at the W band (95 GHz) and D band (130 GHz) is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Spectra of metal complexes in frozen aqueous solutions at approximately physiological concentrations can be obtained either in the presence or absence of protein targets. This method is an improvement over the 1H ENDOR method described previously, which involved the difference ENDOR spectrum of exchangeable protons from spectra taken in H2O and D2O. In addition to exchangeable water protons, the 1H ENDOR method is also sensitive to other exchangeable protons, and it is shown here that this method can overestimate hydration numbers for complexes with exchangeable protons at GdH distances similar to that of the coordinated water, for example, from NH groups. The 17O method does not suffer from this limitation. 17O ENDOR spectroscopy is applied to Gd(III) complexes containing zero, one, or two inner-sphere water molecules. In addition, 13C and 1H ENDOR studies were performed to assess the extent of methanol coordination, since methanol is used to produce a glass in these experiments. Under the experimental conditions used for the hydration number determination (30 mol % methanol), fewer than 15 % of the coordination sites were found to be occupied by methanol.

Chemistry, Physical↗

PubMed2005

Probing the water coordination of protein-targeted MRI contrast agents by pulsed ENDOR spectroscopy.

A novel methodology based on electron-nuclear double resonance (ENDOR) spectroscopy is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Proton ENDOR spectra can be obtained at approximately physiological concentrations for metal complexes in frozen aqueous solutions either in the presence or absence of protein targets. It is shown that, depending on the structure of the co-ligand, the water hydration number of a complex in aqueous solution can be significantly different to when the complex is noncovalently bound to a protein. From the ENDOR spectra of the exchangeable protons, precise information on the metal-proton distance can be derived as well. These essential parameters directly correlate with the efficacy of MRI contrast agents and should therefore aid the development of novel, highly efficient compounds targeted to various proteins.

Contrast Media↗

PubMed2005

When are two waters worse than one? Doubling the hydration number of a Gd-DTPA derivative decreases relaxivity.

The synthesis of a novel ligand, based on N-methyl-diethylenetriaminetetraacetate and containing a diphenylcyclohexyl serum albumin binding group (L1) is described and the coordination chemistry and biophysical properties of its Gd(III) complex Gd-L1 are reported. The Gd(III) complex of the diethylenetriaminepentaacetate analogue of the ligand described here (L2) is the MRI contrast agent MS-325. The effect of converting an acetate to a methyl group on metal-ligand stability, hydration number, water-exchange rate, relaxivity, and binding to the protein human serum albumin (HSA) is explored. The complex Gd-L1 has two coordinated water molecules in solution, that is, [Gd(L1)(H2O)2]2- as shown by D-band proton ENDOR spectroscopy and implied by 1H and 17O NMR relaxation rate measurements. The Gd-H(water) distance of the coordinated waters was found to be identical to that found for Gd-L2, 3.08 A. Loss of the acetate group destabilizes the Gd(III) complex by 1.7 log units (log K(ML) = 20.34) relative to the complex with L2. The affinity of Gd-L1 for HSA is essentially the same as that of Gd-L2. The water-exchange rate of the two coordinated waters on Gd-L1 (k(ex) = 4.4x10(5) s(-1)) is slowed by an order of magnitude relative to Gd-L2. As a result of this slow water-exchange rate, the observed proton relaxivity of Gd-L1 is much lower in a solution of HSA under physiological conditions (r1(obs) = 22.0 mM(-1) s(-1) for 0.1 mM Gd-L1 in 0.67 mM HSA, HEPES buffer, pH 7.4, 35 degrees C at 20 MHz) than that of Gd-L2 (r1(obs) = 41.5 mM(-1) s(-1)) measured under the same conditions. Despite having two exchangeable water molecules, slow water exchange limits the potential efficacy of Gd-L1 as an MRI contrast agent.

Contrast Media↗

PubMed2005

Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin.

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.

Carbon Isotopes↗

PubMed2004

Characterization of protein-ligand interactions by high-resolution solid-state NMR spectroscopy.

A novel approach for detection of ligand binding to a protein in solid samples is described. Hydrated precipitates of the anti-apoptotic protein Bcl-xL show well-resolved (13)C-(13)C 2D solid-state NMR spectra that allow site-specific assignment of resonances for many residues in uniformly (13)C-enriched samples. Binding of a small peptide or drug-like organic molecule leads to changes in the chemical shift of resonances from multiple residues in the protein that can be monitored to characterize binding. Differential chemical shifts can be used to distinguish between direct protein-ligand contacts and small conformational changes of the protein induced by ligand binding. The agreement with prior solution-state NMR results indicates that the binding pocket in solid and liquid samples is similar for this protein. Advantages of different labeling schemes involving selective (13)C enrichment of methyl groups of Ala, Val, Leu, and Ile (Cdelta1) for characterizing protein-ligand interactions are also discussed. It is demonstrated that high-resolution solid-state NMR spectroscopy on uniformly or extensively (13)C-enriched samples has the potential to screen proteins of moderate size ( approximately 20 kDa) for ligand binding as hydrated solids. The results presented here suggest the possibility of using solid-state NMR to study ligand binding in proteins not amenable to solution NMR.

Binding Sites↗

PubMed2003

Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases.

Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated alpha-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1'-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5'-PO(4) (ImmHP) resemble the transition state charge when protonated at 4'-N and bind tightly to these enzymes with K(d) values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4'-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP*ImmH*PO(4) and HGPRTase*ImmHP*MgPP(i) indicate protonation at N-4' for the tightly bound inhibitors. The 1'-(13)C and 1'-(1)H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1'-C toward sp(2) geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4'-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions.

Catalytic Domain↗

PubMed2003

Electron transfer in cyanobacterial photosystem I: I. Physiological and spectroscopic characterization of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX.

The Photosystem I (PS I) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp697PsaA and Trp677PsaB are pi-stacked with the head group of the phylloquinones and are H-bonded to Ser692PsaA and Ser672PsaB, whereas Arg694PsaA and Arg674PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and FX. The mutants W697FPsaA, W677FPsaB, S692CPsaA, S672CPsaB, R694APsaA, and R674APsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P700+A1- radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697FPsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692CPsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P700+A1- and the CW EPR spectra of photoaccumulated A1-. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P700 and A1 similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR.

Binding Sites↗

PubMed2003

Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX.

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.

Binding Sites↗

PubMed2002

Assembly of photosystem I. II. Rubredoxin is required for the in vivo assembly of F(X) in Synechococcus sp. PCC 7002 as shown by optical and EPR spectroscopy.

The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, F(X), F(A), and F(B), are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubA mutant. The flash-induced decay kinetics of both P700(+) in the visible and A(1)- in the near-UV show that charge recombination occurs between P700(+) and A(1)- in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A(1). In agreement, the spin-polarized X-band EPR spectrum of P700(+) A(1)- at low temperature shows that an electron cycle between A(1)- and P700(+) occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [F(A)/F(B)]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to A(1)- center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of F(X), F(B), and F(A), the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the F(X) iron-sulfur cluster but that F(X) is not required for the biosynthesis of trimeric P700-A(1) cores. Since the PsaC protein requires the presence of F(X) for binding, the absence of F(A) and F(B) may be an indirect result of the absence of F(X).

Bacterial Proteins↗