No abstract

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD–RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7 Å resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.

Levels, folding, and function of the infamous cancer and inflammatory disease-related signaling molecule Stat3 are regulated by interaction with the chaperonin TRiC; manipulation of this interaction is a therapeutic avenue for exploration.

The eubacterium Thermus thermophilus uses a macromolecular assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at ∼16 Å resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the V O region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in V O gives insight into the mechanism of torque generation from proton translocation. membrane protein | single particle analysis V acuolar-type ATPases (V-ATPases) in eukaryotes function as ATP-driven proton pumps that acidify intracellular compartments including lysosomes, endosomes, and secretory vesicles. This acidification, in turn, affects diverse processes including protein sorting and degradation, overall ion homeostasis, and protection of cells from oxidative stress (1). Extracellular acidification by V-ATPases is linked to tumor invasion and metastasis and osteoporosis (2). F-type ATP synthases and V-type ATPases are evolutionarily related but differ in the details of subunit composition and arrangement. Both F-and V-type ATPases use a rotary catalytic mechanism where proton translocation through the membranebound F O or V O region, respectively, generates a torque on a rotor subcomplex that drives ATP synthesis in the F 1 or V 1 region. The enzymes can also run in the opposite direction with ATP hydrolysis in the F 1 or V 1 region resulting in proton pumping through F O or V O . This mechanism has been the subject of a large body of research for F-type ATP synthases (e.g., 3-5), but there has also been direct demonstration of rotary catalysis for V-ATPases (6). Some archaea and eubacteria use a complex more closely related to VATPase than F-type ATP synthase, sometimes called an A-ATPase, to generate their supply of ATP (7).The V-ATPase from the eubacterium Thermus thermophilus is composed of nine different subunits with a stoichiometry of A 3 B 3 CDE 2 FG 2 IL 12 (Fig. S1). Subunit nomenclature for this family of enzymes differs between F-and V-type complexes and from organism to organism. Where the eukaryotic ATP synthase F 1 catalytic region consists of α 3 β 3 γδε, the V-ATPase V 1 catalytic region consists of B 3 A 3 DF with no equivalent of the ε-subunit. The catalytic A-subunit of V-ATPase is homologous to the F-type ATP synthas...

No abstract

Polydopamine (PDA)-coated nanoparticles are adhesive bionanomaterials widely utilized in intracellular applications, yet how their adhesiveness affects their colloidal stability and their interactions with serum proteins and mammalian cells remain unclear. In this work, we systematically investigate the combined effects of dopamine (DA) concentration and polymerization time (both reaction parameters spanning 2 orders of magnitude) on the morphological diversity of PDA-coated nanoparticles by coating PDA onto gold nanoparticle cores. Independent of the DA concentration, Au@PDA NPs remain largely aggregated upon several hours of limited polymerization; interestingly, extended polymerization for 2 days or longer yield randomly aggregated NPs, nearly monodisperse NPs, or worm-like NP chains in the ascending order of DA concentration. Upon exposure to serum proteins, the specific type of proteins adsorbed to the Au@PDA NPs strongly depends upon the DA concentration. As DA concentration increases, less albumin and more hemoglobin subunits adhere. Moreover, cellular uptake is a strong function of polymerization time. Serum-stabilized Au@PDA NPs prepared by limited polymerization enter Neuro-2a and HeLa cancer cells more abundantly than those prepared by extended polymerization. Our data underscore the importance of DA concentration and polymerization time for tuning the morphology and degree of intracellular delivery of PDA-coated nanostructures.

No abstract