An Integrated Approach to Unique NMR Assignment of Methionine Methyl Resonances in Proteins.

The application of methyl nuclear magnetic resonance (NMR) spectroscopy in protein side-chain structural studies offers unique advantages of greater peak sensitivity, even for high-molecular-weight proteins. Traditionally, the utility of methyl NMR has often been limited by the difficulty in assigning the methyl resonances. Herein, a mass spectrometry (MS)-assisted strategy to assign the methyl resonances of methionine residues is presented.

Analytical characterization of IgG Fc subclass variants through high-resolution separation combined with multiple LC-MS identification.

With the rapid growth of recombinant monoclonal antibodies and intravenous immunoglobulin (IVIg) medicines, the understanding of human immunoglobulin G (IgG) subclasses becomes more necessary. It is essential to develop effective techniques and methodologies which have the capability for deep characterization. We have created an approach by applying LC and liquid chromatography-mass spectrometry (LC-MS) methods to thoroughly characterize Fc/2 sequence variants for human IgG subclasses in complex samples.

Polarized X-ray absorption spectroscopy of single-crystal Mn(V) complexes relevant to the oxygen-evolving complex of photosystem II.

High-valent Mn-oxo species have been suggested to have a catalytically important role in the water splitting reaction which occurs in the Photosystem II membrane protein. In this study, five- and six-coordinate mononuclear Mn(V) compounds were investigated by polarized X-ray absorption spectroscopy in order to understand the electronic structure and spectroscopic characteristics of high-valent Mn species. Single crystals of the Mn(V)-nitrido and Mn(V)-oxo compounds were aligned along selected molecular vectors with respect to the X-ray polarization vector using X-ray diffraction.

Resonant inelastic X-ray scattering (RIXS) spectroscopy at the Mn K absorption pre-edge-a direct probe of the 3d orbitals.

A study of the Mn K absorption pre-edges in oxides using resonant inelastic X-ray scattering (RIXS) spectroscopy is presented. The energy transfer dimension enhances the separation of the pre-edge (predominantly 1s to 3d transitions) from the main K-edge and a detailed analysis is thus possible. The RIXS spectra are sensitive to the Mn spin state.

The active form of the Saccharomyces cerevisiae ribonucleotide reductase small subunit is a heterodimer in vitro and in vivo.

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y*) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (beta2) and Y4 (beta'2).

EPR distance measurements support a model for long-range radical initiation in E. coli ribonucleotide reductase.

The class I E. coli ribonucleotide reductase, composed of homodimers of R1 and R2, catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The reduction process involves the tyrosyl radical on R2 that generates a transient thiyl radical on R1 over a proposed distance of 35 A.

Structure of the nitrogen-centered radical formed during inactivation of E. coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine-5'-diphosphate: trapping of the 3'-ketonucleotide.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1.

Orientation of calcium in the Mn4Ca cluster of the oxygen-evolving complex determined using polarized strontium EXAFS of photosystem II membranes.

The oxygen-evolving complex of photosystem II (PS II) in green plants and algae contains a cluster of four Mn atoms in the active site, which catalyzes the photoinduced oxidation of water to dioxygen. Along with Mn, calcium and chloride ions are necessary cofactors for proper functioning of the complex. The current study using polarized Sr EXAFS on oriented Sr-reactivated samples shows that Fourier peak II, which fits best to Mn at 3.

The electronic structure of Mn in oxides, coordination complexes, and the oxygen-evolving complex of photosystem II studied by resonant inelastic X-ray scattering.

Resonant inelastic X-ray scattering (RIXS) was used to collect Mn K pre-edge spectra and to study the electronic structure in oxides, molecular coordination complexes, as well as the S1 and S2 states of the oxygen-evolving complex (OEC) of photosystem II (PS II). The RIXS data yield two-dimensional plots that can be interpreted along the incident (absorption) energy or the energy transfer axis. The second energy dimension separates the pre-edge (predominantly 1s to 3d transitions) from the main K-edge, and a detailed analysis is thus possible.

Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms.

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted.

Chloride ligation in inorganic manganese model compounds relevant to photosystem II studied using X-ray absorption spectroscopy.

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn-Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC.

Mn oxidation states in tri- and tetra-nuclear Mn compounds structurally relevant to photosystem II: Mn K-edge X-ray absorption and Kβ X-ray emission spectroscopy studies.

X-Ray spectroscopy is used to examine the effect of the manganese oxidation state for a series of Mn model compounds. Sensitive to Mn oxidation and structural symmetry, X-ray absorption and emission spectroscopy (XAS and XES) provide complementary insights. However, few benchmark examples of complexes with similar structures but in different oxidation states are available to evaluate data from unknown structures like the oxygen evolving complex (OEC) of Photosystem II (PSII).

Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl dadicals in the R2 subunit of the E. coli ribonucleotide reductase.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor.

Calcium EXAFS establishes the Mn-Ca cluster in the oxygen-evolving complex of photosystem II.

The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion.

The Mn cluster in the S(0) state of the oxygen-evolving complex of photosystem II studied by EXAFS spectroscopy: are there three Di-mu-oxo-bridged Mn(2) moieties in the tetranuclear Mn complex?

A key component required for an understanding of the mechanism of the evolution of molecular oxygen by the photosynthetic oxygen-evolving complex (OEC) in photosystem II (PS II) is the knowledge of the structures of the Mn cluster in the OEC in each of its intermediate redox states, or S-states. In this paper, we report the first detailed structural characterization using Mn extended X-ray absorption fine structure (EXAFS) spectroscopy of the Mn cluster of the OEC in the S(0) state, which exists immediately after the release of molecular oxygen. On the basis of the EXAFS spectroscopic results, the most likely interpretation is that one of the di-mu-oxo-bridged Mn-Mn moieties in the OEC has increased in distance from 2.

Strontium EXAFS Reveals the Proximity of Calcium to the Manganese Cluster of Oxygen-Evolving Photosystem II.

The oxygen-evolving complex of Photosystem II (PS II) in green plants and algae contains a cluster of four manganese atoms in the active site, which catalyzes the photoinduced oxidation of water to dioxygen. Along with Mn, calcium and chloride ions are necessary cofactors for proper functioning of the complex. A key unresolved question is whether Ca is close to the Mn cluster, within about 3.