55 research outputs found
Drift Models on Complex Projective Space for Electron-Nuclear Double Resonance
ENDOR spectroscopy is an important tool to determine the complicated
three-dimensional structure of biomolecules and in particular enables
measurements of intramolecular distances. Usually, spectra are determined by
averaging the data matrix, which does not take into account the significant
thermal drifts that occur in the measurement process. In contrast, we present
an asymptotic analysis for the homoscedastic drift model, a pioneering
parametric model that achieves striking model fits in practice and allows both
hypothesis testing and confidence intervals for spectra. The ENDOR spectrum and
an orthogonal component are modeled as an element of complex projective space,
and formulated in the framework of generalized Fr\'echet means. To this end,
two general formulations of strong consistency for set-valued Fr\'echet means
are extended and subsequently applied to the homoscedastic drift model to prove
strong consistency. Building on this, central limit theorems for the ENDOR
spectrum are shown. Furthermore, we extend applicability by taking into account
a phase noise contribution leading to the heteroscedastic drift model. Both
drift models offer improved signal-to-noise ratio over pre-existing models.Comment: 68 pages, 10 figure
High-resolution measurement of long-range distances in RNA: pulse EPR spectroscopy with TEMPO-labeled nucleotides
Structural information at atomic resolution of biomolecular assemblies, such as RNA and RNA protein complexes, is fundamental to comprehend biological function. Modern spectroscopic methods offer exceptional opportunities in this direction. Here we present the capability of pulse EPR to report high-resolution long-range distances in RNAs by means of a recently developed spin labeled nucleotide, which carries the TEMPO group directly attached to the nucleobase and preserves WatsonâCrick base-pairing. In a representative RNA duplex with spin-label separations up to 28 base pairs (â8 nm) we demonstrate that the label allows for a model-free conversion of inter-spin distances into base-pair separation (Îbp) if broad-band pulse excitation at Q band frequencies (34 GHz) is applied. The observed distance distribution increases from Âą0.2 nm for Îbp = 10 to only Âą0.5 nm for Îbp = 28, consistent with only small deviations from the âidealâ A-form RNA structure. Molecular dynamics (MD) simulations conducted at 20 °C show restricted conformational freedom of the label. MD-generated structural deviations from an âidealâ A-RNA geometry help disentangle the contributions of local flexibility of the label and its neighboring nucleobases and global deformations of the RNA double helix to the experimental distance distributions. The study demonstrates that our simple but strategic spin labeling procedure can access detailed structural information on RNAs at atomic resolution over distances that match the size of macromolecular RNA complexes
ENDOR Spectroscopy and DFT Calculations: Evidence for the Hydrogen-Bond Network Within Îą2 in the PCET of E. coli Ribonucleotide Reductase
Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: Îą2 and β2. β2 contains a stable di-iron tyrosyl radical (Y[subscript 122]â˘) cofactor required to generate a thiyl radical (C[subscript 439]â˘) in Îą2 over a distance of 35 Ă
, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y[subscript 122] â W[subscript 48][?] â Y[subscript 356] in β2, across the subunit interface to Y[subscript 731] â Y[subscript 730] â C[subscript 439] in Îą2. Within Îą2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH2Y) replaced Y[subscript 730] in Îą2, and this mutant was incubated with β2, cytidine 5â˛-diphosphate, and adenosine 5â˛-triphosphate to generate a NH2Y730⢠in D2O. [[superscript 2]H]-Electronânuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of Îą2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C[subscript 439] and Y[subscript 731]. A third proton was assigned to a water molecule in close proximity (2.2 Ă
OâH¡¡¡O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH[subscript 2]Y[subscript 730]⢠are consistent with the combined effect of the hydrogen bonds to Cys[subscript 439] and Tyr[subscript 731], both nearly perpendicular to the ring plane of NH[subscript 2]Y[subscript 730]. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in Îą2 of the active RNR complex, for which no structural data are available.National Institutes of Health (U.S.) (NIH GM29595
The CCG-domain-containing subunit SdhE of succinate:quinone oxidoreductase from Sulfolobus solfataricus P2 binds a [4Feâ4S] cluster
In type E succinate:quinone reductase (SQR), subunit SdhE (formerly SdhC) is thought to function as monotopic membrane anchor of the enzyme. SdhE contains two copies of a cysteine-rich sequence motif (CXnCCGXmCXXC), designated as the CCG domain in the Pfam database and conserved in many proteins. On the basis of the spectroscopic characterization of heterologously produced SdhE from Sulfolobus tokodaii, the protein was proposed in a previous study to contain a labile [2Feâ2S] cluster ligated by cysteine residues of the CCG domains. Using UV/vis, electron paramagnetic resonance (EPR), 57Fe electronânuclear double resonance (ENDOR) and MĂśssbauer spectroscopies, we show that after an in vitro cluster reconstitution, SdhE from S. solfataricus P2 contains a [4Feâ4S] cluster in reduced (2+) and oxidized (3+) states. The reduced form of the [4Feâ4S]2+ cluster is diamagnetic. The individual iron sites of the reduced cluster are noticeably heterogeneous and show partial valence localization, which is particularly strong for one unique ferrous site. In contrast, the paramagnetic form of the cluster exhibits a characteristic rhombic EPR signal with gzyx = 2.015, 2.008, and 1.947. This EPR signal is reminiscent of a signal observed previously in intact SQR from S. tokodaii with gzyx = 2.016, 2.00, and 1.957. In addition, zinc K-edge X-ray absorption spectroscopy indicated the presence of an isolated zinc site with an S3(O/N)1 coordination in reconstituted SdhE. Since cysteine residues in SdhE are restricted to the two CCG domains, we conclude that these domains provide the ligands to both the ironâsulfur cluster and the zinc site
Advanced electron paramagnetic resonance on the catalytic ironâsulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase
Understanding Factors Associated With Psychomotor Subtypes of Delirium in Older Inpatients With Dementia
Radical Transfer in E. Coli Ribonucleotide Reductase: a NHâYâââ/RâââA-Îą Mutant Unmasks a New Conformation of the Pathway Residue 731
United States. National Institutes of Health (GM29595
Cross-polarisation ENDOR for spin-1 deuterium nuclei
Efficient transfer of spin polarisation from electron to nuclear spins is emerging as a common target of several advanced spectroscopic experiments, ranging from sensitivity enhancement in nuclear magnetic resonance (NMR) and methods for the detection of single molecules based on optically detected magnetic resonance (ODMR) to hyperfine spectroscopy. Here, we examine the feasibility of electron-nuclear cross-polarisation at a modified Hartmann-Hahn condition (called eNCP) for applications in ENDOR experiments with spin-1 deuterium nuclei, which are important targets in studies of hydrogen bonds in biological systems and materials. We have investigated a two-spin model system of deuterated malonic acid radicals in a single crystal. Energy matching conditions as well as ENDOR signal intensities were determined for a spin Hamiltonian under the effect of microwave and radiofrequency irradiation. The results were compared with numerical simulations and 94-GHz ENDOR experiments. The compelling agreement between theoretical predictions and experimental results demonstrates that spin density operator formalism in conjunction with suitable approximations in regard to spin relaxation provides a satisfactory description of the polarisation transfer effect. The results establish a basis for future numerical optimizations of polarisation transfer experiments using multiple-pulse sequences or shaped pulses and for moving from model systems to real applications in disordered systems
Structure of the tyrosyl biradical in mouse R2 ribonucleotide reductase from high-field PELDOR
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