Organization of intracellular reactions with rationally designed RNA assemblies

The rules of nucleic acid base-pairing have been used to construct nanoscale architectures and organize biomolecules, but little has been done to apply this technology in vivo. We designed and assembled multidimensional RNA structures and used them as scaffolds for the spatial organization of bacterial metabolism. Engineered RNA modules were assembled into discrete, one-dimensional, and two-dimensional scaffolds with distinct protein-docking sites and used to control the spatial organization of a hydrogen-producing pathway. We increased hydrogen output as a function of scaffold architecture. Rationally designed RNA assemblies can thus be used to construct functional architectures in vivo.     

Stable, monomeric variants of lambda Cro obtained by insertion of a designed beta-hairpin sequence

lambda Cro is a dimeric DNA binding protein. Random mutagenesis and a selection for Cro activity have been used to identify the contacts between Cro subunits that are crucial for maintenance of a stably folded structure. To obtain equivalent contacts in a monomeric system, a Cro variant was designed and constructed in which the antiparallel beta-ribbon that forms the dimer interface was replaced by a beta-hairpin. The engineered monomer has a folded structure similar to wild type, is significantly more stable than wild type, and exhibits novel half-operator binding activity.     

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.     

Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A

The zinc finger DNA-binding motif occurs in many proteins that regulate eukaryotic gene expression. The crystal structure of a complex containing the three zinc fingers from Zif268 (a mouse immediate early protein) and a consensus DNA-binding site has been determined at 2.1 angstroms resolution and refined to a crystallographic R factor of 18.2 percent. In this complex, the zinc fingers bind in the major groove of B-DNA and wrap part way around the double helix. Each finger has a similar relation to the DNA and makes its primary contacts in a three-base pair subsite. Residues from the amino-terminal portion of an alpha helix contact the bases, and most of the contracts are made with the guanine-rich strand of the DNA. This structure provides a framework for understanding how zinc fingers recognize DNA and suggests that this motif may provide a useful basis for the design of novel DNA-binding proteins.     

How fast-folding proteins fold

An outstanding challenge in the field of molecular biology has been to understand the process by which proteins fold into their characteristic three-dimensional structures. Here, we report the results of atomic-level molecular dynamics simulations, over periods ranging between 100 μs and 1 ms, that reveal a set of common principles underlying the folding of 12 structurally diverse proteins. In simulations conducted with a single physics-based energy function, the proteins, representing all three major structural classes, spontaneously and repeatedly fold to their experimentally determined native structures. Early in the folding process, the protein backbone adopts a nativelike topology while certain secondary structure elements and a small number of nonlocal contacts form. In most cases, folding follows a single dominant route in which elements of the native structure appear in an order highly correlated with their propensity to form in the unfolded state.     

The role of protein structure in chromatographic behavior

Chromatographic retention is determined by a relatively small number of amino acids located in a chromatographic contact region on the surface of a polypeptide. This region is determined by the mode of separation and the amino acid distribution within the polypeptide. The contact area may be as small as a few hundred square angstroms in bioaffinity chromatography. In contrast, the contact region in ion exchange, reversed phase, hydrophobic interaction and the other nonbioaffinity separation modes is much broader, ranging from one side to the whole external surface of a polypeptide. Furthermore, structural changes that alter the chromatographic contact region will alter chromatographic properties. Thus, although immunosorbents can be very useful in purifying proteins of similar primary structure, they will be ineffective in discriminating between small, random variations within a structure. Nonbioaffinity columns complement affinity columns in probing a much larger portion of solute surface and being able to discriminate between protein variants.     

Polarization of the C. elegans embryo by RhoGAP-mediated exclusion of PAR-6 from cell contacts

Early embryos of some metazoans polarize radially to facilitate critical patterning events such as gastrulation and asymmetric cell division; however, little is known about how radial polarity is established. Early embryos of Caenorhabditis elegans polarize radially when cell contacts restrict the polarity protein PAR-6 to contact-free cell surfaces, where PAR-6 regulates gastrulation movements. We have identified a Rho guanosine triphosphatase activating protein (RhoGAP), PAC-1, which mediates C. elegans radial polarity and gastrulation by excluding PAR-6 from contacted cell surfaces. We show that PAC-1 is recruited to cell contacts, and we suggest that PAC-1 controls radial polarity by restricting active CDC-42 to contact-free surfaces, where CDC-42 binds and recruits PAR-6. Thus, PAC-1 provides a dynamic molecular link between cell contacts and PAR proteins that polarizes embryos radially.     

Prevalence and patterns of same-gender sexual contact among men

The prevalence and patterns of same-gender sexual contact among men are key components of models of the spread of HIV infection and AIDS in the U.S. population. Previous estimates by Kinsey et al. from data collected between 1938 and 1948 have been widely criticized for inadequacies of sample design. New lower-bound estimates of prevalence developed from data from a national sample survey conducted in 1970 indicate that minimums of 20.3 percent of adult men in the United States in 1970 had sexual contact to orgasm with another man at some time in life; 6.7 percent had such contact after age 19; and between 1.6 and 2.0 percent had such contact within the previous year. Although these estimates incorporate adjustments for missing data, the likelihood of underreporting suggests that these estimates might be lower bounds on the prevalence of same-gender sex among men. Two sets of alternative estimates are derived to assess the sensitivity of these estimates to the assumptions made in imputing values to missing data. Detailed estimates are presented by frequency of contact, age, education, and marital status; and supporting estimates are derived from a 1988 national survey. Data from both the 1970 and 1988 surveys indicate that never-married men are more likely than other men to have had same-gender sexual contacts within the last year. The 1970 survey also indicates, however, that approximately half the men estimated to have such contacts are found among the more numerous population of currently or previously married men.     

X-ray diffraction from magnetically oriented solutions of macromolecular assemblies

A simple system was developed for obtaining x-ray diffraction patterns from magnetically oriented solutions of macromolecular assemblies. A small permanent magnet was designed that produces a magnetic field of 16 kilogauss in a volume of 1 cubic millimeter and is mountable on most x-ray cameras. Many subcellular structures have sufficient diamagnetic anisotropy that they exhibit orientation in dilute solution when placed between the poles of the magnet. Diffraction from solutions oriented in this magnet can provide substantially more structural information than small-angle scattering from isotropic solutions. In favorable cases, such as dilute solutions of filamentous bacteriophages, it is possible to produce oriented fiber diffraction patterns from which intensities along layer lines can be measured to 7-angstrom resolution. The magnetically induced birefringence observed in solutions of other macromolecular assemblies suggests that this technique may have broad applicability to subcellular structures.     

Membrane structure: some general principles

The arrangement of lipids and some proteins in the erythrocyte membrane has been discussed. The conclusions from this are listed here as a set of general guidelines for the structure of membranes of higher organisms: some of these rules may be wrong. But at this stage it seems useful to sharpen our thoughts in this way and thereby focus attention on various specific points. 1) The basis of a membrane is a lipid bilayer with (i) choline phospholipids and glycolipids in the external half and (ii) amino (and possibly some choline) phospholipids in the cytoplasmic half. There is effectively no lipid exchange across the bilayer (unless enzymatically catalyzed) (68). 2) Some proteins extend across the bilayer. Where this is so, they will in general have carbohydrate on their surface remote from the cytoplasm. This carbohydrate may prevent the protein diffusing out of the membrane into the cytoplasm; it acts as a lock on the protein. 3) Just as lipids do not flip-flop, proteins do not rotate across the membrane. Lateral motion or rotation of lipids and proteins in the plane of the bilayer may be expected. 4) Most membrane protein is associated with the inner, cytoplasmic, urface of the membrane. Proteins are not usually associated exclusively with the outer half of the lipid bilayer. 5) Membrane proteins are a special class of cytoplasmic proteins, not of secreted proteins.     

High-probability uniquantal transmission at excitatory synapses in barrel cortex

The number of vesicles released at excitatory synapses and the number of release sites per synaptic connection are key determinants of information processing in the cortex, yet they remain uncertain. Here we show that the number of functional release sites and the number of anatomically identified synaptic contacts are equal at connections between spiny stellate and pyramidal cells in rat barrel cortex. Moreover, our results indicate that the amount of transmitter released per synaptic contact is independent of release probability and the intrinsic release probability is high. These properties suggest that connections between layer 4 and layer 2/3 are tuned for reliable transmission of spatially distributed, timing-based signals.     

The energetic basis of specificity in the Eco RI endonuclease--DNA interaction

High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence.     

植物表达重组蛋白的N-糖基化研究进展

分子农业中,植物作为生物反应器生产药用重组蛋白是近十几年来基因工程研究的一大热点。植物具有完整的真核表达系统、重组蛋白通常可以正确组装、折叠和修饰,同时,由于植物表达体系的方便、廉价等优点,利用转基因植物生产药用蛋白的研究迅猛发展,已有100余种蛋白中得到表达,有些已进入商业化生产。但是植物与动物对蛋白的转译后修饰并不是完全保守的,植物是否能够真正“忠实”地表达出实用、可靠的目的蛋白产品已成为备受瞩目的话题,由于大多数药用蛋白都是分泌型蛋白,因此植物表达重组蛋白的N-糖基化成为新的研究热点。笔者对植物表达体系对药用蛋白的N-糖基化修饰途径和人源化改造植物表达重组蛋白的研究进展两个方面加以综述,并对生物反应器的未来发展方向进行了探讨和展望。     

Evidence for macromolecular protein rings in the absence of bulk water

We have examined the architecture of a protein complex in the absence of bulk water. By determining collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topology of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addition of a specific RNA molecule increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility separation for defining shapes of heterogeneous macromolecular assemblies.     

Computational design of self-assembling protein nanomaterials with atomic level accuracy

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.     

Connectivity patterns of crayfish giant interneurons: visualization of synaptic regions with cobalt dye

Intracellular injection of cobalt dye was used to visualize electrical synapses between two pairs of central giant interneurons and giant motoneurons in the crayfish central nervous system. A pair of giant motoneurons in each ganglion contacts the interneurons, but not all contact points are functional synapses. Cobalt dye reveals numerous fine projections that are present at synaptic contact points and absent at nonsynaptic contacts; intracellular recording confirms this correlation. The different connectivity patterns of the two pairs of interneurons are consistent with the different behavior patterns which they evoke.     

Characterization of a helical protein designed from first principles

The question of how the primary amino acid sequence of a protein determines its three-dimensional structure is still unanswered. One approach to this problem involves the de novo design of model peptides and proteins that should adopt desired three-dimensional structures. A systematic approach was aimed at the design of a four-helix bundle protein. The gene encoding the designed protein was synthesized and the protein was expressed in Escherichia coli and purified to homogeneity. The protein was shown to be monomeric, highly helical, and very stable to denaturation by guanidine hydrochloride (GuHCl). Thus a globular protein has been designed that is capable of adopting a stable, folded structure in aqueous solution.     

Imaging spin transport in lateral ferromagnet/semiconductor structures

We directly imaged electrical spin injection and accumulation in the gallium arsenide channel of lateral spin-transport devices, which have ferromagnetic source and drain tunnel-barrier contacts. The emission of spins from the source was observed, and a region of spin accumulation was imaged near the ferromagnetic drain contact. Both injected and accumulated spins have the same orientation (antiparallel to the contact magnetization), and we show that the accumulated spin polarization flows away from the drain (against the net electron current), indicating that electron spins are polarized by reflection from the ferromagnetic drain contact. The electrical conductance can be modulated by controlling the spin orientation of optically injected electrons flowing through the drain.