Mechanism of Pyrroloquinoline Quinone-Dependent Hydride Transfer Chemistry from Spectroscopic and High-Resolution X-ray Structural Studies of the Methanol Dehydrogenase from Methylococcus capsulatus (Bath).

The active site of methanol dehydrogenase (MDH) contains a rare disulfide bridge between adjacent cysteine residues. As a vicinal disulfide, the structure is highly strained, suggesting it might work together with the pyrroloquinoline quinone (PQQ) prosthetic group and the Ca2+ ion in the catalytic turnover during methanol (CH3OH) oxidation. We purify MDH from Methylococcus capsulatus (Bath) with the disulfide bridge broken into two thiols. Spectroscopic and high-resolution X-ray crystallographic studies of this form of MDH indicate that the disulfide bridge is redox active. We observe an internal redox process within the holo-MDH that produces a disulfide radical anion concomitant with a companion PQQ radical, as evidenced by an optical absorption at 408 nm and a magnetically dipolar-coupled biradical in the EPR spectrum. These observations are corroborated by electron-density changes between the two cysteine sulfurs of the disulfide bridge as well as between the bound Ca2+ ion and the O5-C5 bond of the PQQ in the high-resolution X-ray structure. On the basis of these findings, we propose a mechanism for the controlled redistribution of the two electrons during hydride transfer from the CH3OH in the alcohol oxidation without formation of the reduced PQQ ethenediol, a biradical mechanism that allows for possible recovery of the hydride for transfer to an external NAD+ oxidant in the regeneration of the PQQ cofactor for multiple catalytic turnovers. In support of this mechanism, a steady-state level of the disulfide radical anion is observed during turnover of the MDH in the presence of CH3OH and NAD+.

Delineating the membrane-disrupting and seeding properties of the TDP-43 amyloidogenic core.

The amyloidogenic core in the TAR DNA-binding protein (TDP-43) C-terminal fragment has been characterized with its chemical, biochemical, and structural properties delineated. Various properties of the core sequence, including membrane impairment ability and the seeding effect, have also been studied.

Cotranslational protein folding within the ribosome tunnel influences trigger-factor recruitment.

In recent years, various folding zones within the ribosome tunnel have been identified and explored through x-ray, cryo-electron microscopy (cryo-EM), and molecular biology studies. Here, we generated ribosome-bound nascent polypeptide complexes (RNCs) with different polyalanine (poly-A) inserts or signal peptides from membrane/secretory proteins to explore the influence of nascent chain compaction in the Escherichia coli ribosome tunnel on chaperone recruitment. By employing time-resolved fluorescence resonance energy transfer and immunoblotting, we were able to show that the poly-A inserts embedded in the passage tunnel can form a compacted structure (presumably helix) and reduce the recruitment of Trigger Factor (TF) when the helical motif is located in the region near the tunnel exit. Similar experiments on nascent chains containing signal sequences that may form compacted structural motifs within the ribosome tunnel and lure the signal recognition particle (SRP) to the ribosome, provided additional evidence that short, compacted nascent chains interfere with TF binding. These findings shed light on the possible controlling mechanism of nascent chains within the tunnel that leads to chaperone recruitment, as well as the function of L23, the ribosomal protein that serves as docking sites for both TF and SRP, in cotranslational protein targeting.

The interplay of turn formation and hydrophobic interactions on the early kinetic events in protein folding.

While both turn formation and hydrophobic interactions play dominant roles in the initiation of protein folding, their individual contributions to the folding kinetics and to the structural stability of the protein still remain poorly understood. Here, we applied a photolabile linker to "cage" some important structural motifs, including both α-helices and ß-sheets, into their non-native states. These "caged" structural motifs are then relaxed by laser-flash photolysis and their refolding events followed by photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD). These experiments, combined with our previous results, revealed that spontaneous α-helix formation can occur extremely rapidly (10(8)-10(9) s(-1)) if the process is driven solely by turn formation followed by helix propagation. However, if sequestering of the side chains of hydrophobic amino acid residues participates in the refolding process, which may provide additional driving force beyond that afforded by turn formation alone, the refolding rate will be retarded, often by many orders of magnitude. This is usually the case in the formation of three-stranded ß-sheets (10(7)-10(8) s(-1)) and ß-hairpins (10(5)-10(6) s(-1)). Thus, we propose that proteins take advantage of the hierarchy of timescales associated with either turn formation, hydrophobic interactions, or global collapse of tertiary structure to accomplish the folding process in an orderly fashion, as these events are sufficiently separated in time and do not interfere with one another.

Dioscorea phytocompounds enhance murine splenocyte proliferation ex vivo and improve regeneration of bone marrow cells in vivo.

Specific cytokines have been tested clinically for immunotherapy of cancers; however, cytotoxicity has often impaired their usefulness. Consequently, alternative approaches are increasingly desirable. Dioscorea spp. tuber is a widely used traditional Chinese medicinal herb claimed to confer immunostimulatory activity. In this study, we evaluated Dioscorea as an adjuvant therapy for use alongside chemotherapy for cancer. Phytocompounds from Dioscorea tubers were ethanol fractioned and used for ex vivo splenocyte proliferation assay or in vivo force-feeding of mice pre-treated with the chemotherapy agent 5-fluorouracil. Co-treatment with a 50-75% ethanol-partitioned fraction of the tuber extract of D. batatas (DsCE-II) and interleukin (IL)-2 resulted in a significantly higher rate of murine splenocyte cell proliferation ex vivo than treatment with DsCE-II or IL-2 alone. This DsCE-II fraction, which contains a polysaccharide with a high proportion of ß-1,4-linkage mannose (≥64%), also promoted the regeneration of specific progenitor cell populations in damaged bone marrow tissues of 5-fluorouracil-treated mice. Colony-forming unit (CFU) analyses demonstrated that the population of CFU-GM cells, but not CFU-GEMM or BFU-E cells, preferentially recovered to ~67% in the bone marrow of immune-suppressed mice fed with DsCE-II. DsCE-II efficacy level was ~85% of that obtained by subcutaneous administration of recombinant G-CSF proteins (5 µg kg(-1)) in mice tested in parallel. This study suggests that the DsCE-II fraction of D. batatas extract may be considered for further development as a dietary supplement for use alongside chemotherapy during cancer treatment.

Crystal structure of Adenylylsulfate reductase from Desulfovibrio gigas suggests a potential self-regulation mechanism involving the C terminus of the beta-subunit.

Adenylylsulfate reductase (adenosine 5'-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-A resolution. Different from the alpha(2)beta(2)-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six alphabeta-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the alpha-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the alpha-helix (amino acid 289 to 299) in the alpha-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the beta-subunit wraps around the alpha-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the alpha-subunit from another alphabeta-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the alpha-subunit and Asp159 in the C terminus of the beta-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the beta-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the beta-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the beta-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the beta-subunit.

Tuning the conformational properties of the prion peptide.

Previously, we disclosed that O-linked glycosylation of Ser-132 or Ser-135 could dramatically change the amyloidogenic property of the hamster prion peptide (sequence 108-144). This peptide, which corresponds to the flexible loop and the first beta-strand in the structure of the prion protein, is a random coil when it is initially dissolved in buffer, but amyloid fibrils are formed with time. Thus, it offers a convenient model system to observe and compare how different chemical modifications and sequence mutations alter the amyloidogenic property of the peptide within a reasonable experimental time frame. In our earlier study, aside from uncovering a site-specificity of the glycosylation on the fibrillogenesis, different effects of alpha-GalNAc and beta-GlcNAc were observed. In this work, we explore further how different sugar configurations affect the conformational property of the polypeptide chain. We compare the effects of O-linked glycosylation by the common sugars alpha-GalNAc, beta-GlcNAc with their non-native analogs beta-GalNAc, alpha-GlcNAc in an effort to uncover the origin of the sugar-specificity on the fibril formation. We find that the anomeric configuration of the sugar is the most important factor affecting the fibrillogenesis. Sugars with the glycosidic bond in the alpha-configuration at Ser-135 have a dramatic inhibitory effect on the structural conversion of the glycosylated peptide. Because O-glycosylation of Ser-135 with alpha-linked sugars also promote the formation of three slowly converting conformations at the site of glycosylation, we surmise that the amyloidogenic property of the peptide is related to its conformational flexibility, and the proclivity of this region of the peptide to undergo the structural conversion from the random coil to form the beta-structure. Upon O-glycosylation with an alpha-linked sugar, this conversion is inhibited and the nucleation of fibril formation is largely retarded. Consistent with this scenario, Arg-136 is the residue most affected in the TOCSY NMR spectra of the glycosylated peptides, other than the serine site modified. In addition, when Arg-136 is substituted by Gly, a mutation that should provide higher structural flexibility in this part of the peptide, the amyloidogenic property of the peptide is greatly enhanced, and the inhibition effect of glycosylation is largely diminished. These results are consistent with Ser-135 and Arg-136 being part of the kink region involved in the structural conversion.

A proposed role for Leishmania major carboxypeptidase in peptide catabolism.

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.

Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS.

Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.

Synthesis, structure and dioxygen reactivity of a bis(micro-iodo)dicopper(I) complex supported by the [N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine ligand.

The air-sensitive bis(micro-iodo)dicopper(I) complex 1 supported by [N-(3,5-di-tert-butyl-2-hydroxybenzyl)-N,N-di-(2-pyridylmethyl)]amine (L) has been prepared by treating copper(I) iodide with L in anhydrous THF. Compound 1 crystallizes as a dimer in space group C2/c. Each copper(I) center has distorted tetrahedral N2I2 coordination geometry with Cu-N(pyridyl) distances 2.061(3) and 2.063(3) A, Cu-I distances 2.6162(5) and 2.7817(5) and a Cu...Cu distance of 2.9086(8) A. Complex 1 is rapidly oxidized by dioxygen in CH2Cl2 with a 1 : 1 stoichiometry giving the bis(micro-iodo)peroxodicopper(II) complex [Cu(L)(micro-I)]2O2 (2). The reaction of 1 with dioxygen has been characterized by UV-vis, mass spectrometry, EPR and Cu K-edge X-ray absorption spectroscopy at low temperature (193 K) and above. The mass spectrometry and low temperature EPR measurements suggested an equilibrium between the bis(micro-iodo)peroxodicopper(II) complex 2 and its dimer, namely, the tetranuclear (peroxodicopper(II))2 complex [Cu(L)(micro-I)]4O4 (2'). Complex 2 undergoes an effective oxo-transfer reaction converting PPh3 into O=PPh3 under anaerobic conditions. At sufficiently high concentration of PPh3, the oxygen atom transfer from 2 to PPh3 was followed by the formation of [Cu(PPh3)3I]. The dioxygen reactivity of 1 was compared with that known for other halo(amine)copper(I) dimers.

Tuning the conformation properties of a peptide by glycosylation and phosphorylation.

We have deployed the alpha-helical hairpin peptide (alpha-helix/turn/alpha-helix) and used it as a model system to explore how glycosylation and phosphorylation might affect the conformational properties of the peptide. The native conformations of the modified peptides in buffer solution have been compared with that of the wild-type peptide by nuclear magnetic resonance spectroscopy. Circular dichroism spectroscopy was used to probe the effects of an O-linked beta-GlcNAc and a phosphate group on the overall folding stability of the peptide. Finally, the rate of fibrillogenesis was used to infer the effects of these chemical modifications on the alpha-to-beta transition as well as the rate of nucleation of amyloidogenesis.

Quantitative proteomic analysis of metabolic regulation by copper ions in Methylococcus capsulatus (Bath).

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them.

A molecular dynamics method for calculating molecular volume changes appropriate for biomolecular simulation.

Photothermal methods permit measurement of molecular volume changes of solvated molecules over nanosecond timescales. Such experiments are an important tool in investigating complex biophysical phenomena including identifying transient species in solution. Developing a microscopic understanding of the origin of volume changes in the condensed phase is needed to complement the experimental measurements. A molecular dynamics (MD) method exploiting available simulation methodology is demonstrated here that both mimics experimental measurements and provides microscopic resolution to the thermodynamic measurements. To calculate thermodynamic volume changes over time, isothermal-isobaric (NPT) MD is performed on a solution for a chosen length of time and the volume of the system is thus established. A further simulation is then performed by "plucking" out a solute molecule of interest to determine the volume of the system in its absence. The difference between these volumes is the thermodynamic volume of the solute molecule. NPT MD allows the volume of the system to fluctuate over time and this results in a statistical uncertainty in volumes that are calculated. It is found in the systems investigated here that simulations lasting a few nanoseconds can discern volume changes of approximately 1.0 ml/mole. This precision is comparable to that achieved empirically, making the experimental and theoretical techniques synergistic. The technique is demonstrated here on model systems including neat water, both charged and neutral aqueous methane, and an aqueous beta-sheet peptide.