Protein structure determination using long-distance constraints from double-quantum coherence ESR: study of T4 lysozyme

We report the use of a novel pulsed ESR technique for distance measurement, based on the detection of double quantum coherence (DQC), which yields high quality dipolar spectra, to significantly extend the range of measurable distances in proteins using nitroxide spin-labels. Eight T4 lysozyme (T4L) mutants, doubly labeled with methanethiosulfonate spin-label (MTSSL), have been studied using DQC-ESR at 9 and 17 GHz. The distances span the range from 20 A for the 65/76 mutant to 47 A for the 61/135 mutant. The high quality of the dipolar spectra also allows the determination of the distance distributions, the width of which can be used to set upper and lower bounds in future computational strategy. It is also demonstrated that the shape of these distributions can reveal the presence of multiple conformations of the spin-label, an issue of critical relevance to the structural interpretation of the distances. The distances and distributions found in this study are readily rationalized in terms of the known crystal structure, the characteristic conformers of the nitroxide side chains, and molecular modeling. This study sets the stage for the use of DQC-ESR for determining the tertiary structure of large proteins with just a small number of long-distance constraints.     

Calculation of Double-Quantum-Coherence Two-dimensional Spectra: Distance Measurements and Orientational Correlations

The double-quantum-coherence (DQC) echo signal for two coupled nitroxides separated by distances ?10 Å, is calculated rigorously for the six-pulse sequence. Successive application of six pulses on the initial density matrix, with appropriate inter-pulse time evolution and coherence pathway selection leaves only the coherent pathways of interest. The amplitude of the echo signal following the last π pulse can be used to obtain a one-dimensional (1D) dipolar spectrum (Pake doublet), and the echo envelope can be used to construct the 2D DQC spectrum. The calculations are carried out using the product space spanned by the two electron-spin magnetic quantum numbers m ₁, m ₂ and the two nuclear-spin magnetic quantum numbers M ₁, M ₂, describing, e.g. two coupled nitroxides in bilabeled proteins. The density matrix is subjected to a cascade of unitary transformations taking into account dipolar and electron exchange interactions during each pulse and during the evolution in the absence of a pulse. The unitary transformations use the eigensystem of the effective spin Hamiltonians obtained by numerical matrix diagonalization. Simulations are carried out for a range of dipolar interactions, D, and microwave magnetic field strength B ₁ for both fixed and random orientations of the two ¹⁴N (and ¹⁵N) nitroxides. Relaxation effects were not included. Several examples of 1D and 2D Fourier transforms of the time-domain signals versus dipolar evolution and spin-echo envelope time variables are shown for illustration. Comparisons are made between 1D rigorous simulations and analytical approximations. The rigorous simulations presented here provide insights into DQC electron spin resonance spectroscopy, they serve as a standard to evaluate the results of approximate theories, and they can be employed to plan future DQC experiments.     

Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles

We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that alphaS does not retain a "hairpin" structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.     

Inter-helix distances in lysophospholipid micelle-bound alpha-synuclein from pulsed ESR measurements

We demonstrate the use of pulsed ESR spectroscopy to measure intramolecular distances in the Parkinson's disease-associated protein alpha-synuclein bound to detergent and lysophospholipid micelles. We show that the inter-helical separation between the two helices formed upon binding to micelles is dependent on micelle composition, with micelles formed from longer acyl chains leading to an increased splaying of the two helices. Our data suggest that the topology of alpha-synuclein is not strongly constrained by the linker region between the two helices and instead depends on the geometry of the surface to which the protein is bound.     

The Effect of Support Nature and the Temperature of Preliminary Calcination on the Atomic Catalytic Activity of Supported Palladium Catalysts for the Complete Oxidation of Hydrocarbons

It is shown that for palladium catalysts supported on various supports, there is no effect of thermal activation consisting of an increase in the turnover number (TON) of palladium with an increase in the temperature of sample calcination from 500 to 700°C, as was observed in the case of similar supported platinum catalysts; for two palladium–alumina catalysts calcined at 500°C and differing in the concentration of palladium and dispersity, the TON of low-dispersity palladium is substantially higher than the TON of high-dispersity palladium; all other conditions being the same, the TON of supported palladium is determined by the support nature.     

Effect of Thermal Activation of Supported Catalysts Pt/MeOx,Where MOx = Al2O3, CeO2, La2O3, and ZrO2, for Complete Oxidation

A sharp increase in the atomic catalytic activity (ACA) of supported platinum catalysts in the model reaction of n-pentane complete oxidation is found on going from the preliminary calcination temperature of 500–600°C to a temperature of 700°C. ACA increases by an order of magnitude for the Pt/-Al₂O₃ system, 3 times for Pt/ZrO₂, and 1.5 times for Pt/CeO₂. The per-gram activities of all catalysts decrease because of a decrease in the dispersion of supported platinum with an increase in the temperature of preliminary calcination.     

Multifrequency Two-Dimensional Fourier Transform ESR: An X/Ku–Band Spectrometer

A two-dimensional Fourier Transform ESR (2D FT ESR) spectrometer operating at 9.25 and 17.35 GHz is described. The Ku-band bridge uses an efficient heterodyne technique wherein 9.25 GHz is the intermediate frequency. At Ku-band the sensitivity is increased by almost an order of magnitude. One may routinely collect a full 2D ELDOR spectrum in less than 20 min for a sample containing 0.5–5 nmol of nitroxide spin-probe in the slow-motional regime. Broad spectral coverage at Ku-band is obtained by use of a bridged loop-gap resonator (BLGR) and of a dielectric ring resonator (DR). It is shown that an even more uniform spectral excitation is obtained by using shorter microwave pulses of about 3 ns duration. The dead-time at Ku-band is just 30–40 ns, yielding an improved SNR in 2D ELDOR spectra of nitroxide spin-probes withT₂as short as 20–30 ns. A comparison of 2D ELDOR spectra obtained at 9.25 and 17.35 GHz for spin-labeled phospholipid probes (16PC) in 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) membrane vesicles showed that both spectra could be satisfactorily simulated using the same set of model parameters even though they are markedly different in appearance. The improved sensitivity and shorter dead-time at Ku-band made it possible to obtain orientation-dependent 2D ELDOR spectra of the Cholestane (CSL) spin-probe in macroscopically aligned lipid bilayers of egg yolk PC using samples containing only 1 mg of lipid and just 5 nmol of spin-probe.