Distances between the b-subunits in the tether domain of F0F1-ATP synthase from E. coli

The arrangement of the b-subunits in the holo-enzyme F₀F₁-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F₀F₁-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The hydrophilic F₁-part and the hydrophobic membrane-integrated F₀-part are connected by a central and a peripheral stalk. The peripheral stalk consists of two b-subunits. Cysteine mutations are introduced in the tether domain of the b-subunit at b-40, b-51, b-53, b-62 or b-64 and labeled with a nitroxide spin label. Conventional (9 GHz), high-field (95 GHz) and pulsed EPR spectroscopy reveal: All residues are in a relatively polar environment, with mobilities consistent with helix sites. The distance between the spin labels at each b-subunit is 2.9 nm in each mutant, revealing a parallel arrangement of the two helices. They can be in-register but separated by a large distance (1.9 nm), or at close contact and displaced along the helix axes by maximally 2.7 nm, which excludes an in-register coiled-coil model suggested previously for the b-subunit. Binding of the non-hydrolysable nucleotide AMPPNP to the spin-labeled enzyme had no significant influence on the distances compared to that in the absence of nucleotides.     

Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2

We have used a bifunctional spin label (BSL) to cross-link Cys⁷⁰⁷ (SH1) and Cys⁶⁹⁷ (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A·M·ADP·P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A·M·ADP. In contrast, helical order is observed for the strongly bound state A·M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A·M·ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A·M·ADP, slow rotational mobility comparable to that of A·M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.     

Inferred motions of the S3a helix during voltage-dependent K+ channel gating

The gating of voltage-dependent potassium channels is controlled by conformational changes in voltage sensor domains. Previous studies have shown that the S1 and the S2 helices of the voltage sensor are static with respect to motion across the membrane, while the voltage sensor paddle consisting of the C-terminal half of S3 (S3b) and the charge-bearing S4 is mobile. The mobile component is attached to S1 and S2 via the S2-S3 turn and the N-terminal half of S3 (S3a). In this study, we analyze KvAP, an archaebacterial voltage-dependent potassium channel, to study the mobility with respect to translation across the membrane of S3a. We utilize an assay based on attachment of tethered biotin and its site-specific accessibility to avidin. Our results reveal that the S3a helix does not move appreciably across the membrane in association with gating. The static behavior of S3a constrains the conformations available to the voltage sensor when it closes and suggests that a set of negative countercharges within the membrane's inner leaflet remains intact in the closed conformation.     

Structural modeling of dual-affinity purified Pho84 phosphate transporter

The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.     

GABAA receptor transmembrane amino acids are critical for alcohol action: disulfide cross‐linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites

Alcohols and inhaled anesthetics modulate GABA_A receptor (GABA_AR) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter‐ or intra‐subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA‐mediated currents in wild‐type and mutant α1β2γ2 GABA_ARs expressed in Xenopus laevis oocytes. We tested for: (i) inter‐subunit cross‐linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of β2TM2 and TM3; (ii) intra‐subunit cross‐linking: a cysteine located either in β2TM1 [β2(T225C)] or in TM2 [β2(N265C)] was paired with a cysteine in different locations along β2TM3. Three inter‐subunit cysteine pairs and four intra‐subunits cross‐linked. In three intra‐subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra‐subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter‐ or intra‐subunit sites is determined by the specific receptor and ligand.

     

Mapping of Drebrin Binding Site on F-Actin

Drebrin is a filament-binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full-length protein. Site-directed mutagenesis, electron microscopic reconstruction, and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and MTS1 [1,1-methanediyl bis(methanethiosulfonate)]. Site-directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1 and that native cysteine 308 of drebrin is near C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, can link the N-terminal G-S extension of the recombinant DrABD to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different lengths (5.4-19 Å). These results suggest that the “core” DrABD is centered on actin subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of electron microscopic reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin-binding proteins.     

F-Actin Structure Destabilization and DNase I Binding Loop Fluctuations: Mutational Cross-Linking and Electron Microscopy Analysis of Loop States and Effects on F-Actin

The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues > 45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (> 41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.     

Identification of a stable flavin-thiolate adduct in heterotetrameric sarcosine oxidase

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing>or=90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.     

The Role of Prefibrillar Structures in the Assembly of a Peptide Amyloid

The self-assembly of proteins into stable, fibrillar aggregates is a general property of polypeptides most notably associated with degenerative diseases termed amyloidoses. These nano- to micrometer scale structures are formed predominantly of β-sheets that self-assemble by a nucleation-dependent mechanism. The rate-limiting step of assembly involves stabilization of high-energy intermediates in a kinetic step termed nucleation. Determination of the structural characteristics of these high-energy intermediates has been elusive, as its members are the least populated states on the assembly pathway. Using a peptide derived from diabetes-related amyloid, we use electron paramagnetic resonance (EPR) spectroscopy and disulfide crosslinking to show that fibers are composed of parallel, in-register β-sheets. Kinetic studies are then used to infer the structural elements of the pre-nucleation intermediates. Notably, stabilization of this ensemble is shown to depend on the number but not the position of amide side chains within the primary sequence. Additionally, fiber formation is accelerated by constructs that mimic the intra-sheet structure of the fiber. Our data suggest that pre-nucleation intermediates sample intra- β-sheet structure and place bounds on the possible nucleation mechanisms for fiber assembly. Understanding the nucleation of fibrillogenesis is critical so that this process can be prevented in disease and productively controlled by design.     

Quantitative proteomic analysis of lignocellulolytic enzymes by Phanerochaete chrysosporium on different lignocellulosic biomass

Lignocellulosic biomass from agricultural crop residues and forest waste represents an abundant renewable resource for bioenergy and future biofuel. The current bottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar. To understand the enzymatic hydrolysis of complex biomasses, in this report, lignocellulolytic enzymes secretion by Phanerochaete chrysosporium cultivated in different natural lignocellulosic biomass such as corn stover, hay, sawdust, sugarcane baggase, wheat bran and wood chips were quantitatively analyzed with the iTRAQ technique using LC-MS/MS. A diverse groups of enzymes, including cellulases, glycoside hydrolases, hemicellulases, lignin degrading enzymes, peroxidases, esterases, lipases, chitinases, peptidases, protein translocating transporter and hypothetical proteins were quantified, of which several were novel lignocellulosic biomass hydrolyzing enzymes. The quantitative expression and regulation of lignocellulolytic enzymes by P. chrysosporium were dependent on the nature and complexity of lignocellulosic biomass as well as physical size of the biomass. The iTRAQ data revealed oxidative and hydrolytic lignin degrading mechanism of P. chrysosporium. Numerous proteins presumed to be involved in natural lignocellulosic biomass transformation and degradation were expressed and produced in variable quantities in response to different agricultural and forest wastes.