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PubMed1992

Self-assembly into fibrils of a homotrimer of type I collagen.

Type I collagen, the most abundant structural protein in vertebrates, is comprised of two alpha 1(I) chains and one alpha 2(I) chain. Fibroblasts from a proband with osteogenesis imperfecta, however, were shown to synthesize a type I procollagen that was a homotrimer of pro alpha 1(I) chains. The absence of pro alpha 2(I) chains in the procollagen provided a unique opportunity to assess the role of the alpha 2(I) chain in collagen fibrillogenesis by examining the self-assembly de novo of the homotrimeric collagen generated in vitro. The results demonstrated that the fibrils formed by the homotrimeric collagen had an asymmetric banding pattern similar to fibrils of normal heterotrimeric type I collagen. However, the efficiency for self-assembly of the homotrimer into fibrils was markedly reduced in that the critical concentration at 37 degrees C was 40-fold greater than for self-assembly of the heterotrimeric molecule. A van't Hoff-type plot of the data was used to determine values for delta G, delta H and delta S. The values indicated the self-assembly of the homotrimer is similar to self-assembly of the heterotrimer in that the process is entropy driven. The process is, however, less favorable in that the delta G value was 10 kJ/mol less negative. The results suggest that the presence of the alpha 2(I) chain in type I collagen helps drive the self-assembly process, probably because the alpha 2(I) chain is more hydrophobic than the alpha 1(I) chain and, therefore, smaller amounts of structured water may be lost during self-assembly of the homotrimer than during self-assembly of the heterotrimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed2002

Assembly-dependent trafficking assays in the detection of receptor-receptor interactions.

Assembly-dependent trafficking is a property of many multimeric membrane protein complexes; this coupling of assembly and trafficking processes provides an important cellular quality control mechanism, ensuring that only properly folded and assembled complexes are expressed on the cell surface. In all membrane protein complexes whose trafficking is known to be assembly-dependent, at least one of the subunits contains an endoplasmic reticulum (ER) retention/retrieval signal that is shielded on subunit assembly, allowing the assembled protein complex to traffic to the plasma membrane. Under these conditions, presence of the normally retained subunit on the cell surface can be used as an indirect index of protein assembly in the ER. In this article, I describe the design of two complementary approaches (trafficking enhancement and trap assays) that can be used separately or in combination to determine whether two (or more) proteins assemble in the ER, i.e., whether they constitutively oligomerize. Both of the approaches are based on the measurement of plasma membrane-expressed proteins using antibody-mediated detection of extracellularly expressed epitopes and subsequent luminometric quantification. These methods provide a straightforward and relatively inexpensive way to assess protein-protein interactions early in the synthetic pathway.

Animals↗
PubMed1992

Bis-ANS as a specific inhibitor for microtubule-associated protein induced assembly of tubulin.

5,5'-Bis[8-(phenylamino)-1-naphthalenesulfonate] (bis-ANS), the fluorescent probe which binds to tubulin, inhibits its assembly into microtubules [Horowitz et al. (1984) J. Biol. Chem. 259, 14647-14650]. The results described in this paper demonstrate that bis-ANS is quite distinct from other well-known microtubule inhibitors in its specificity of action. The inhibitory potentials of bis-ANS and its three structural analogues ANS, Prodan [6-propionyl-2-(dimethylamino)naphthalene], and NSA (naphthalenesulfonic acid) have been compared. It is found that they can be arranged in the following order according to their polymerization inhibitory potentials: bis-ANS approximately equal to Prodan much greater than ANS greater than NSA. Interestingly, the naphthalene nucleus is sufficient to cause inhibition of polymerization. Detailed experiments were carried out to examine the mode of assembly inhibition by aminonaphthalenes at the molecular level, using bis-ANS as a representative. It was found that there was little or no effect of bis-ANS on the assembly of tubulin when polymerization was induced by assembly promoters like taxol, DMSO, or glutamate, or on the assembly of subtilisin-digested protein (tubulin S), for all of which half-maximal inhibition could not be achieved even at 120 microM bis-ANS. On the contrary, bis-ANS acts as an inhibitor in the case of MAP- (MAP2 and tau) and poly(L-lysine)-induced assembly of tubulin, with half-maximal inhibitory concentrations ranging from 1.5 to 7.6 microM. Our results place bis-ANS as a novel inhibitor, which seems to specifically inhibit C-termini-mediated assembly. Of all assembly inhibitors known so far, none exhibits such selection.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1985

Pressure effects on actin self-assembly: interspecific differences in the equilibrium and kinetics of the G to F transformation.

Purified skeletal muscle actins from species whose ambient pressures range from 1 to greater than 500 atm were examined for the sensitivity to hydrostatic pressure of the globular (G) to filamentous (F) self-assembly reaction. Both the equilibrium position and the kinetics of self-assembly were affected by pressure. Increased pressure shifted the self-assembly equilibrium toward the monomer (G) state and reduced the rate of F-actin assembly. For most of the actins studied, the perturbation by pressure of F-actin formation decreased with increasing measurement of pressure, indicating that F-actin has a higher compressibility than G-actin. The increase in system volume and compressibility concomitant with the assembly of F-actin can be interpreted as reflections of the major role played by hydrophobic effects in stabilizing F-actin and of the existence of "hard" binding sites, in the terminology of Torgerson et al. [Torgerson, P. M., Drickamer, H. G., & Weber, G. (1979) Biochemistry 18, 3079-3083], in the actin subunits. For actin from the deepest occurring species studied, the teleost fish Coryphaenoides armatus, which occurs to depths of approximately 5000 m (equivalent to 501 atm of pressure), there was no difference in compressibility between G-actin and F-actin; that is, the effect of increasing pressure on self-assembly was linear over the entire pressure range examined, 600 atm. The self-assembly reaction of the actin from C. armatus also differed from that of the other actins examined in that the G to F equilibrium was relatively insensitive to increased pressure; i.e., the volume change (delta V) of assembly was small.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1987

GTP hydrolysis during microtubule assembly.

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.

Animals↗
PubMed1981

Electrostatic contributions to the energetics of dimer-tetramer assembly in human hemoglobin: pH dependence and effect of specifically bound chloride ions.

The pH dependence and effects of specifically bound chloride ions on the electrostatic contribution to the energetics of human hemoglobin dimer-tetramer assembly were computed for deoxy- and liganded hemoglobin. In the absence of bound chloride, the electrostatic contribution models the observed contrasting pH dependence of dimer-tetramer assembly for deoxy- and oxyhemoglobin. The effect of specifically bound chloride on the computations depends on the number and placement of the anions. Deoxy assembly shows a greater sensitivity to anion binding, with effects propagating as far as 32 A from the binding site. This sensitivity suggests a mechanism for electronic communication with the heme. At pH 7.4, 24-34% of the experimental value for deoxy and 73-85% for oxy dimer-tetramer assembly stabilization are predicted. Together with the findings of Chu and Ackers [Chu, A. H., & Ackers, G. K. (1981) J. Biol. Chem. 256, 1199] and other recent work, these results suggest that salt bridge formation is not the dominant energetic factor favoring deoxyhemoglobin dimer-tetramer assembly. Results of this work suggest that the marked electrostatic stabilization favoring oxy dimer-tetramer assembly may be a significant contributor to the quaternary enhancement observed in assembly reactions whereas the nonelectrostatic factors favoring deoxy dimer-tetramer assembly may be largely responsible for quaternary constraint.

Binding Sites↗
PubMed2005

Conformational dynamics of amyloid beta-protein assembly probed using intrinsic fluorescence.

Formation of toxic oligomeric and fibrillar structures by the amyloid beta-protein (Abeta) is linked to Alzheimer's disease (AD). To facilitate the targeting and design of assembly inhibitors, intrinsic fluorescence was used to probe assembly-dependent changes in Abeta conformation. To do so, Tyr was substituted in Abeta40 or Abeta42 at position 1, 10 (wild type), 20, 30, 40, or 42. Fluorescence then was monitored periodically during peptide monomer folding and assembly. Electron microscopy revealed that all peptides assembled readily into amyloid fibrils. Conformational differences between Abeta40 and Abeta42 were observed in the central hydrophobic cluster (CHC) region, Leu17-Ala21. Tyr20 was partially quenched in unassembled Abeta40 but displayed a significant and rapid increase in intensity coincident with the maturation of an oligomeric, alpha-helix-containing intermediate into amyloid fibrils. This process was not observed during Abeta42 assembly, during which small decreases in fluorescence intensity were observed in the CHC. These data suggest that the structure of the CHC in Abeta42 is relatively constant within unassembled peptide and during the self-association process. Solvent accessibility of the Tyr ring was studied using a mixed solvent (dimethyl sulfoxide/water) system. [Tyr40]Abeta40, [Tyr30]Abeta42, and [Tyr42]Abeta42 all were relatively shielded from solvent. Analysis of the assembly dependence of the site-specific intrinsic fluorescence data suggests that the CHC is particularly important in controlling Abeta40 assembly, whereas the C-terminus plays the more significant role in Abeta42 assembly.

Alzheimer Disease↗
PubMed1996

Cooperativity mutants of bacteriophage lambda cI repressor: temperature dependence of self-assembly.

Analytical ultracentrifugation was used to study higher order self-assembly of lambda cI repressors, including eight mutants whose monomer to dimer reactions were recently characterized [Burz et al. (1994) Biochemistry 33, 8399]. Six of the mutants were found to remain dimeric up to 50 microM total protein; the remaining mutants (EK102 and PT158) were found to undergo higher order oligomerization, as does wild-type cI. For these three repressors, we determined the stoichiometries and energetics of higher order assembly over the temperature range 5-40 degrees C. Weak dimerization exhibited by two other mutants, SN228 and SR228, was also evaluated by sedimentation equilibrium over this same temperature range. The end-state for higher order assembly of wild-type cI was determined to be octameric, in agreement with Senear et al. [(1993) Biochemistry 32, 6179-6189]. The assembly free energies resolved by the sedimentation analysis program NONLIN [Johnson, M. L., et al. (1981) Biophys. J. 36, 575-588] leads to the prediction that tetramers may contribute significantly to the intermediate populations during assembly. This analysis of the species populations is in accord with recent conclusions from fluorescence anisotropy studies [Banik et al. (1993) J. Biol. Chem. 268, 3938]. It was found that two of the mutant repressors (EK102 and PTI58) assemble into octamers, but with differing possible intermediates. PT158 satisfies the stoichiometry 8M1 <--> 2M4 <--> M8, while the EK102 data conforms to a 4M2 <--> 2M4 <--> M8 model, similar to WT (both the EK102 and WT data could also be described by a dimer-octamer model with no intermediates). Of the six repressors found in this study to remain dimeric, three exhibit non-cooperative DNA binding (GD147, KN192, YH210), two express intermediate cooperativity (EK188, SR228), and one is fully cooperative (SN228). The three octamerizing repressors are fully cooperative [Burz & Ackers (1994) Biochemistry 33, 8399], suggesting a correlation between their ability to form higher order assemblies and to engage in cooperative DNA binding. Linear van't Hoff plots were obtained for overall assembly of wild-type and EK102 dimers, while that of PT158 monomers was curved, indicating a negative heat capacity change. The van't Hoff analyses of dimerization constants for SN228 and SR228 were distinctly different from each other and also from that of wild type; such differences might be related to the disparate cooperative behavior found previously for these mutants (Burz & Ackers, 1994).

Centrifugation, Isopycnic↗
PubMed1996

Assembly mechanism of Dictyostelium myosin II: regulation by K+, Mg2+, and actin filaments.

Regulated assembly of myosin II in Dictyostelium discoideum amoebae partially controls the orderly formation of contractile structures during cytokinesis and cell migration. Kinetic and structural analyses show that Dictyostelium myosin II assembles by a sequential process of slow nucleation and controlled growth that differs in rate and mechanism from other conventional myosins. Nuclei form by an ordered progression from myosin monomers to parallel dimers to 0.43 microns long antiparallel tetramers. Lateral addition of dimers to bipolar tetramers completes the assembly of short (0.45 microns) blunt-ended thick filaments. Myosin heads are not staggered along the length of tapered thick filaments as in skeletal muscle, nor are bipolar minifilaments formed as in Acanthamoeba. The overall assembly reaction incorporating both nucleation and growth could be kinetically characterized by a second-order rate constant (kobs,N+G) of 1.85 x 10(4) M-1 s-1. Individual rate constants obtained for nucleation, kobs,N = 4.5 x 10(3) M-1 s-1, and growth, kobs,G = 2.5 x 10(4) M-1 s-1, showed Dictyostelium myosin II to be the slowest assembling myosin analyzed to date. Nucleation and growth stages were independently regulated by Mg2+, K+, and actin filaments. Increasing concentrations of K+ from 50 to 150 mM specifically inhibited lateral growth of dimers off nuclei. Intracellular concentrations of Mg2+ (1 mM) accelerated nucleation but maintained distinct nucleation and growth phase kinetics. Networks of actin filaments also accelerated the nucleation stage of assembly, mechanistically accounting for spontaneous formation of actomyosin contractile fibers via myosin assembly (Mahajan et al., 1989). The distinct assembly mechanism and regulation utilized by Dictyostelium myosin II demonstrates that myosins from smooth muscle, striated muscle, and two types of amoebae form unique thick filaments by different pathways.

Actins↗
PubMed1997

Allosteric interactions between DNA strands and monovalent cations in DNA quadruplex assembly: thermodynamic evidence for three linked association pathways.

The series of cooperative transitions that lead to [d(TG4)4.(K+)m] quadruplex assembly upon rapid addition of KCl to d(TG4) strands were studied. Quadruplex samples were dialyzed against KCl then Li-EDTA and found to retain between three and five strongly bound potassiums with affinities >10(6) M-2. Absorbance thermal denaturation (melt) and circular dichroism (CD) equilibrium binding data were obtained. The latter were analyzed using two classes of binding models to simulate the effects of the assumed intermolecular interactions on the binding curves (isotherms). The melt experiments yielded equilibrium dissociation constants (Kd) ranging from 10(-11) to 10(-12) M3 at the melting temperatures. Extrapolating these values to 23 degrees C predicts Kd values in the 10(-28) M3 range if the heat capacity (Cp) is not strongly dependent upon temperature changes over this range. Assuming Ka is equal to 1/Kd (from melting analyses), very large association free energies stabilize the quadruplex at 23 degrees C in 100 mM KCl (DeltaGa = -43 kcal mol-1). Plots of the differential melt curve peak half-widths, a measure of cooperativity, versus d(TG4) concentration showed that quadruplex dissociation is much more cooperative at 400 mM KCl than at 100 mM KCl. Forty-eight hour quadruplex assembly time courses were monitored by CD at 264 nm. Equilibrium quadruplex accumulation generally required over 10 h, and net reaction extents were in the 10-85% range. Hill plots of the data show that initial steps in the multistep pathway are positively cooperative, presumably due to strong strand-cation and strand-strand binding interactions in duplex and triplex assembly reactions, then negatively cooperative in quadruplex formation. Models were developed to rationalize the experimental observations in terms of consecutive cooperative allosteric transitions from cation-deficient relaxed (R) strand-aggregates to cation-containing tense (T) structures, driven by the allosteric effector K+. Quantitative mappings of positive and then negative cooperativity were obtained by fitting the results as a function of strand number incorporated during quadruplex assembly. Surprisingly, models for reactions involving incorporation of five and six strands fit the data better than models involving only four strands. The 5-step "induced fit" model fits the data as well as or better than 3- and 4-step models and better than all of the strand aggregation models that were devised and investigated. Net association free energies (summation operatori=1,n) ranged from -20 to -26 kcal mol-1, approximately half the magnitude of the apparent stabilities measured by absorbance melts. Likely explanations for this discrepancy involve hysteresis and errors due to inadequate equilibration in the melt experiments. Hysteresis is thought to be produced by irreversibility due to different predominant mechanisms in absorbance (dissociation) and CD (association) experiments. The kinetic block to quadruplex assembly can be unambiguously attributed to quadruplex formation and not intermediate steps in the assembly mechanism. On the basis of these results we propose that, in addition to the more conventional assembly mechanisms involving duplex dimerization and stepwise strand addition, quadruplex formation can also proceed by triplex-triplex disproportionation. Interaction statistics arguments that support the energetic feasibility of the disproportionation pathway are presented. The allosteric quadruplex assembly model provides a mechanism which could be used by the cell to simultaneously modulate DNA structure and activity within telomeres, transcriptional promoters, recombination-prone chromatin, and other G-rich DNAs. As a result of this allosterism, cation and strand availability and strand-pairing capabilities could profoundly influence the functional capacity of a particular strand over a relatively narrow range of effector concentration changes. (ABSTRACT TRUNCATED)

Allosteric Regulation↗
PubMed1999

Assembly dynamics of the nucleocapsid shell subunit (P8) of bacteriophage phi6.

Phi6 is an enveloped dsRNA bacteriophage of Pseudomonas syringae. The viral envelope encloses a nucleocapsid, consisting of an RNA-dependent RNA polymerase complex within an icosahedral shell assembled from approximately 800 copies of a 16 kDa subunit (protein P8, encoded by viral gene 8). During infection, the nucleocapsid penetrates the host plasma membrane and enters the cytosol, whereupon the P8 shell disassembles and the polymerase complex is activated. To understand the molecular mechanisms of shell assembly and disassembly-processes that have counterparts in most viral infections-we have investigated the structure, stability, and dynamics of P8 in different assembly states using time-resolved Raman spectroscopy and hydrogen-isotope exchange. In the presence of Ca(2+), which promotes shell assembly, the highly alpha-helical conformation of the P8 subunit is stabilized by rapid assembly into shell-like structures. However, in the absence of Ca(2+), the P8 subunit is thermolabile and unstable, manifested by a slow alpha-helix --> beta-strand conformational change and the accumulation of aberrant aggregates. In both properly assembled shells and aberrant aggregates, the P8 subunit retains an alpha-helical core that is protected against deuterium exchange of amide NH groups. Surprisingly, no additional protection against amide exchange is conferred by the shell lattice. Time-resolved assembly and disassembly experiments in deuterated buffers indicate that the regions of P8 involved in subunit/subunit interactions in the intact shell undergo rapid exchanges, presumably due to local unfolding events that are characterized by low activation barriers. Such localized dynamics of P8 within the shell lattice may mediate the nucleocapsid/host membrane interactions that are required in the cytosol for particle assembly during maturation and disassembly during infection.

Bacteriophage phi 6↗
PubMed2006

Accurate length control of supramolecular oligomerization: Vernier assemblies.

Linear oligomeric supramolecular assemblies of defined length have been generated using the Vernier principle. Two molecules, containing a different number (n and m) of mutually complementary binding sites, separated by the same distance, interact with each other to form an assembly of length (n x m). The assembly grows in the same way as simple supramolecular polymers, but at a molecular stop signal, when the binding sites come into register, the assembly terminates giving an oligomer of defined length. This strategy has been realized using tin and zinc porphyrin oligomers as the molecular building blocks. In the presence of isonicotinic acid, a zinc porphyrin trimer and a tin porphyrin dimer form a 3:4 triple stranded Vernier assembly six porphyrins long. The triple strand Vernier architecture introduced here adds an additional level of cooperativity, yielding a stability and selectivity that cannot be achieved via a simple Vernier approach. The assembly properties of the system were characterized using fluorescence titrations and size-exclusion chromatography (SEC). Assembly of the Vernier complex is efficient at micromolar concentrations in nonpolar solvents, and under more competitive conditions, a variety of fragmentation assemblies can be detected, allowing determination of the stability constants for this system and detailed speciation profiles to be constructed.

Isonicotinic Acids↗
PubMed2006

Plasmon coupling in nanorod assemblies: optical absorption, discrete dipole approximation simulation, and exciton-coupling model.

The shape anisotropy of nanorods gives rise to two distinct orientational modes by which nanorods can be assembled, i.e., end-to-end and side-by-side, analogous to the well-known H and J aggregation in organic chromophores. Optical absorption spectra of gold nanorods have earlier been observed to show a red-shift of the longitudinal plasmon band for the end-to-end linkage of nanorods, resulting from the plasmon coupling between neighboring nanoparticles, similar to the assembly of gold nanospheres. We observe, however, that side-by-side linkage of nanorods in solution shows a blue-shift of the longitudinal plasmon band and a red-shift of the transverse plasmon band. Optical spectra calculated using the discrete dipole approximation method were used to simulate plasmon coupling in assembled nanorod dimers. The longitudinal plasmon band is found to shift to lower energies for end-to-end assembly, but a shift to higher energies is found for the side-by-side orientation, in agreement with the optical absorption experiments. The strength of plasmon coupling was seen to increase with decreasing internanorod distance and an increase in the number of interacting nanorods. For both side-by-side and end-to-end assemblies, the strength of the longitudinal plasmon coupling increases with increasing nanorod aspect ratio as a result of the increasing dipole moment of the longitudinal plasmon. For both the side-by-side and end-to-end orientation, the simulation of a dimer of nanorods having dissimilar aspect ratios showed a longitudinal plasmon resonance with both a blue-shifted and a red-shifted component, as a result of symmetry breaking. A similar result is observed for a pair of similar aspect ratio nanorods assembled in a nonparallel orientation. The internanorod plasmon coupling scheme concluded from the experimental results and simulations is found to be qualitatively consistent with the molecular exciton coupling theory, which has been used to describe the optical spectra of H and J aggregates of organic molecules. The coupled nanorod plasmons are also suggested to be electromagnetic analogues of molecular orbitals. Investigation of the plasmon coupling in assembled nanorods is important for the characterization of optical excitations and plasmon propagation in these nanostructures. The surface plasmon resonance shift resulting from nanorod assembly also offers a promising alternative for analyte-sensing assays.

Absorption↗
PubMed2005

Assembly of multilayer films from polyelectrolytes containing weak and strong acid moieties.

Multilayer films were assembled from a copolymer containing both weakly and strongly charged pendant groups, poly(4-styrenesulfonic acid-co-maleic acid) (PSSMA), deposited in alternation with poly(allylamine hydrochloride) (PAH). The strongly charged groups (styrene sulfonate, SS) are expected to form electrostatic linkages (to enhance film stability), while the weakly charged groups (maleic acid, MA) can alter multilayer film properties because they are responsive to external pH changes. In this study, we varied several assembly conditions such as pH, SS/MA ratio in PSSMA, and the ionic strength of the polyelectrolyte solutions. The multilayer films were also treated by immersion into pH 2 and 11 solutions after assembly. Quartz crystal microgravimetry and UV-visible spectrophotometry showed that the thickness of PSSMA/PAH multilayers decreases with increasing assembly pH regardless of whether salt was present in the polyelectrolyte solutions. When no salt was added, the multilayers are thinner, smoother, and grow less regularly. Atomic force microscopy images indicate that the presence of salt in polyelectrolyte solutions results in rougher surface morphologies, and this effect is especially significant in multilayers assembled at pH 2 and pH 11. When both polyelectrolytes are adsorbed at conditions where they are highly charged, salt was necessary to promote regular multilayer growth. Fourier transform infrared spectroscopy studies show that the carboxylic acids in the multilayers are essentially ionized when assembled from different pHs in 0.5 M sodium chloride solutions, whereas some carboxylic acids remain protonated in the multilayers assembled from solutions with no added salt. This resulted in different pH stability regimes when the multilayers were exposed to different pH solutions, post assembly.

Electrolytes↗
PubMed2003

Productivity-biodiversity relationships depend on the history of community assembly.

Identification of the causes of productivity-species diversity relationships remains a central topic of ecological research. Different relations have been attributed to the influence of disturbance, consumers, niche specialization and spatial scale. One unexplored cause is the history of community assembly, the partly stochastic sequential arrival of species from a regional pool of potential community members. The sequence of species arrival can greatly affect community structure. If assembly sequence interacts with productivity to influence diversity, different sequences can contribute to variation in productivity-diversity relationships. Here we report a test of this hypothesis by assembling aquatic microbial communities at five productivity levels using four assembly sequences. About 30 generations after assembly, productivity-diversity relationships took various forms, including a positive, a hump-shaped, a U-shaped and a non-significant pattern, depending on assembly sequence. This variation resulted from idiosyncratic joint effects of assembly sequence, productivity and species identity on species abundances. We suggest that the history of community assembly should be added to the growing list of factors that influence productivity-biodiversity patterns.

Bacterial Physiological Phenomena↗
PubMed2001

An Eulerian path approach to DNA fragment assembly.

For the last 20 years, fragment assembly in DNA sequencing followed the "overlap-layout-consensus" paradigm that is used in all currently available assembly tools. Although this approach proved useful in assembling clones, it faces difficulties in genomic shotgun assembly. We abandon the classical "overlap-layout-consensus" approach in favor of a new euler algorithm that, for the first time, resolves the 20-year-old "repeat problem" in fragment assembly. Our main result is the reduction of the fragment assembly to a variation of the classical Eulerian path problem that allows one to generate accurate solutions of large-scale sequencing problems. euler, in contrast to the celera assembler, does not mask such repeats but uses them instead as a powerful fragment assembly tool.

Algorithms↗
PubMed2003

Nuclear actin and protein 4.1: essential interactions during nuclear assembly in vitro.

Structural protein 4.1, which has crucial interactions within the spectrin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher-resolution detergent-extracted cell whole-mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

Actins↗
PubMed2003

Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides.

We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (i). gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii). ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii). assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule. We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification. PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements. The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp. However, fully infectious X174 virions were recovered after electroporation into Escherichia coli. Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes. One such isolate had exactly the intended sequence. We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; approximately 60 such segments would be required for a minimal cellular genome.

Bacteriophage phi X 174↗