Critical Concentration For Polymerization Research Articles

Unraveling temperature-dependent supramolecular polymorphism of naphthalimide-substituted benzene-1,3,5-tricarboxamide derivatives

Temperature plays a crucial role in regulating polymorphism in supramolecular polymers. Understanding the mechanism behind temperature-dependent supramolecular polymorphism is crucial as it provides an opportunity to tailor polymorphs for specific properties and applications. In this study, we present our findings on a naphthalimide-substituted benzene-1,3,5-tricarboxamide derivative, R-Nap-1, which exhibits two distinct polymerization pathways at varying temperatures. At 313 K, polymerization results in the formation of an M-chiral polymorph, whereas at 253 K, a P-chiral polymorph is formed. Both polymorphs are notably stable, remaining unchanged for over six months under ambient conditions. Theoretical calculations and experimental investigations allowed us to elucidate the mechanisms underlying these polymorphic transformations. The formation of the M-chiral polymorph at 313 K is attributed to the nucleation and growth of R-Nap-1 monomers once their concentration surpasses a critical threshold. Conversely, at lower temperatures (e.g., 253 K), the monomers undergo facile transformation into dimers due to a lower energy barrier and reduced Gibbs energy compared to the monomeric state. Subsequently, these dimers undergo nucleation-elongation to form the P-chiral polymorph when their concentration exceeds the critical polymerization concentration. The stability and lack of interconversion between the two polymorphs can be attributed to their close thermodynamic stabilities, as evidenced by variable-temperature CD spectra and DFT calculations. These findings highlight the importance of accurate temperature control in supramolecular polymerization processes, making a significant contribution to the understanding of supramolecular polymorphism, thus advancing the field of supramolecular chemistry.

Supramolecular polymerization behavior of a ditopic self-folding biscavitand

Abstract Reported herein is the supramolecular polymerization of a mixture of a feet-to-feet connected biscavitand and a homoditopic quinuclidinium guest that is regulated by cooperativity in the host–guest association. Diffusion-ordered NMR spectroscopy (DOSY) was used to evaluate the supramolecular polymerization in toluene, CHCl3, and tetrahydrofuran (THF). Upon concentrating the solutions of the biscavitand with the quinuclidinium guest in CHCl3 and THF, the diffusion coefficient (D) values were meaningfully decreased, indicating that the host–guest complexation facilitated supramolecular polymerization. In contrast, the slight change of the D value in toluene suggests that supramolecular polymerization was suppressed, although the binding constant (K) between the cavitand and quinuclidinium guest was reported to be 105 L mol−1 in toluene. The viscosity measurements showed both the critical polymerization concentration (CPC) and entangled concentration (Ce) upon concentrating the CHCl3 solution of the mixture. Neither the CPC nor Ce was seen in the toluene solution of the mixture. Accordingly, the strong negative cooperativity in the 1:2 host–guest complexation of the biscavitand discouraged the supramolecular polymerization in toluene. These findings are valuable in deepening the understanding of host–guest association-driven supramolecular polymerization behaviors regulated by a combination of cooperativity and K value in solution.

Synthesis and gas transport properties of hyperbranched network polyimides derived from Tris(4-aminophenyl)benzene

A series of carbon molecular sieve (CMS) membranes have been prepared for CO2/N2 separation based on the aromatic hyperbranched polyimides (HBPIs) synthesized by polymerizing 1,3,5-Tris(4-aminophenyl)benzene (TAPB) and three commercially available dianhydrides, including 1,2,4,5-benzenetetracarboxylic anhydride (PMDA), 2,3,3′,4′-Biphenyltetracarboxylic dianhydride (BPDA), 4,4′-(hexafluoroisopropylidene) diphthalic anhydride (6FDA). The polymerizations were carried out at −60 °C to avoid the gelation at the critical polymerization concentration and the free-standing membranes were successfully obtained from the HBPI solution using directly solvent casting method after post thermal annealing. The hyperbranched molecules swollen, interpenetrated into the adjacent molecules and finally formed a network, when the temperature increased to 60 °C. Both the molecular entanglement and the post thermal imidization contribute to the membrane formation, without resorting to any additional crosslinking agents. The factors governing the gas separation of the HBPI membranes and the CMS ones were investigated using TGA analysis, FT-IR spectroscopy, X-ray diffraction and Raman characterization. The results confirm that the inter-chain spacing in the HBPIs provide the pathways for gas molecules crossing the membrane. The gas permeabilities of the CMS membranes have been further improved owing to the ultramicro- and micro-pores generated after pyrolyzing at 550 °C. For example, the CO2 permeability of XS13-550 significantly increased from 26.0 barrer (XS13) to 16564.4 barrer with a loss of CO2/N2 selectivity from 34.7 (XS13) to 16.6. This work provides a low-temperature process to prepare HBPI precursors without any crosslinking agents.

The Use of Electroactive Ureidopyrimidones As Redox-Responsive Chain Terminators for Supramolecular Polymers

Unlike conventional polymers, in which monomers are reacted to form strong covalent connections, supramolecular polymers are formed by linking together monomers via weaker, non-covalent interactions such as H-bonds, ion-dipole interactions and so on. One very well-studied class of supramolecular polymers are based on the ureidopyrimidones, UPy’s, which dimerize via 4 strong, linear H-bonds in non-competitive solvents. Monomers for supramolecular polymerization can be made by linking together 2 or more UPy units with short covalent chains. Under dimerization conditions, the UPy’s link up to form longer polymeric chains. One of the useful attributes of such polymers is their self-healing ability. Application of heat or mechanical stress will readily break the monomers apart at the H-bonds, but, upon cooling or relief of stress, the bonds reform. The goal of this research is to see if electron transfer can be used as an alternative, more selective stimulus to break apart the UPy chains. As a first step in this direction, the use of our previously studied electroactive UPy’s, 1a and 1b, are being investigated as redox-active chain terminators. For these studies the known UPy monomer, 2, was synthesized. Viscosity measurements of solutions of 2 in 0.1 M NBu4PF6/CH2Cl2 give a critical polymerization concentration of 20 mM. Based on this, 60 mM 2 was chosen as the working concentration for initial experiments giving a solution with a specific viscosity of 3.0 mPa∙s. Upon addition of 2 mM of the ferrocene containing UPy 1a, the viscosity dropped to 1.1 mPa∙s, indicating that 1a is an effective chain terminator, decreasing the lengths of the chains which results in a decrease in the viscosity of the solution The ferrocenes were then completely oxidized to the ferrocenium form in a two compartment electrochemical cell. This caused the viscosity of the solution to increase from 1.0 to 1.7 mPa∙s, suggesting decreased effectiveness of the ferrocenium form as a chain terminator. This is consistent with poorer binding of the ferrocenium form to the bis-UPy. Importantly, reduction back to the ferrocene led to a decrease in the viscosity back to close to where it was initially. In this presentation, the results of further studies on the ability of both 1a and 1b to act as redox-responsive chain terminators for UPy-based supramolecular polymers will be reported. Figure 1

Signalosome nucleation enables digital innate immune proinflammatory responses

Pathogen sensing in innate immune cells begins with the activation of Pathogen Recognition Receptors (PRR) that result in the formation of polymeric protein self‐assemblies known as signalosomes. The proinflammatory response resulting from signalosome formation is thought to produce a binary all‐or‐none response. How this is controlled remains unclear. Because the activation of ordered self‐assemblies like signalosomes implies a rate‐limiting nucleation step, we hypothesized that a nucleation barrier imposed by the signalosome assembly enables the digital all‐or‐none response. Here we focused on the signalosome CARD9‐Bcl10‐MALT1 (CBM), which functions downstream of fungal PRR, Dectin‐1, which activates NF‐κB. We employed DAmFRET, a recently developed method that allows to determine the frequency of protein nucleation in cells using flow cytometry. We determined that CARD9 and Bcl10 form nucleation‐limited polymers in cells with critical concentrations similar to those from in vitro studies. We then dissected the nucleation steps required for signalosome formation. Co‐expression of artificial CARD9 polymer seeds with Bcl10 resulted in the complete nucleation of Bcl10 monomeric protein. Remarkably, CARD9 seeds containing mutations that impair antifungal signaling in humans were unable to nucleate Bcl10, suggesting that a direct seeding effect of CARD9 over Bcl10 is critical for its function. We next asked whether the formation of the signalosome drives the digital activation of NF‐κB. To do so, we coupled a transcriptional fluorescent NF‐κB reporter to DAmFRET. We determined that cells containing Bcl10 assemblies strongly induced NF‐κB activation, while cells expressing a similar protein concentration in the monomeric state did not. We asked next whether mutations that alter the nucleation barriers likewise modulate NF‐κB activation. Disruption of polymer formation with mutation E53R as well as increasing the critical concentration of polymerization with mutant R36E, both impaired NF‐κB activation. In contrast, mutation R58G decreased the nucleation barrier while also exacerbating the activation of NF‐κB. Our results indicate a critical role of nucleation barriers in governing the functional outcome of signalosomes. We anticipate the impact of our work could uncover the molecular mechanism of innate immune digital proinflammatory activation, which may serve as a basis to understand the cause of autoinflammatory diseases.Support or Funding InformationStowers Institute for Medical Research

Multi-functional regulator MapZ controls both positioning and timing of FtsZ polymerization.

The tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.

Linear Supramolecular Polymers Driven by Anion–Anion Dimerization of Difunctional Phosphonate Monomers Inside Cyanostar Macrocycles

Supramolecular polymers have enabled far-reaching fundamental science and the development of diverse macromolecular technologies owing to the reversible and noncovalent chemical connectivities that define their properties. Despite the unabated development of these materials using highly tailorable recognition elements, anion-based polymers remain rare as a result of the weak interactions they mediate. Here, we use design rules inspired by cation-driven polymers to demonstrate a new noncovalent link based on receptor-stabilized anion-anion interactions that enables the efficient linear polymerization of simple difunctional phosphonates. The linear main chain connectivity and molecular topology were confirmed by single crystal X-ray diffraction, which demonstrates the rare 2:2 stoichiometry between the anionic phosphonate end groups and a pair of π-stacked cyanostar macrocycles. The stability of these links enables rapid polymerization of difunctional phosphonates employing different aliphatic linkers (C6H12, C8H16, C10H20, C12H24). Diphosphonates with greater chain flexibility (C12H24) enable greater polymerization with an average degree of polymerization of nine emerging at 10 mM. Viscosity measurements show a transition from oligomers to polymers at the critical polymerization concentration of 5 mM. In a rare correlation, NMR spectroscopy shows a coincident molecular signature of the polymerization at 5 mM. These polymers are highly concentration dependent, reversibly polymerize with acid and base, and respond to competitive anions. They display the design simplicity of metallo-supramolecular polymers with transfer of the strong 2:2 recognition chemistry to macromolecules. The simplicity and understanding of this new class of supramolecular polymer is anticipated to open opportunities in tailoring anion-based functional materials.

Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration.

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1–2 μM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.

Cross-linked supramolecular polymers synthesized by photo-initiated thiol-ene click reaction of supramonomers

Supramonomers refer to monomers fabricated via noncovalent interactions and can be polymerized through conventional covalent methods. In this article, we realized the fabrication of cross-linked supramolecular polymers through photo-initiated thiol-ene click reaction of quadruple hydrogen bonded supramonomers. It is found that the cross-linked supramolecular polymer has a higher critical polymerization concentration than the linear supramolecular polymer, because the orientation of the trifunctional monomer leads to cyclize at low concentrations. It is anticipated that this research can enrich the methodology of fabricating cross-linked supramolecular polymers and provide a new phenomenon to understand the self-assembling behavior of cross-linked supramolecular polymers.

Tubulin Monomer-Monomer Association is Less Influenced by the Solvent than Dimer-Dimer Association: Structure and Function of Tubulin Interaction Interfaces

The ab-tubulin heterodimer is the basic unit of microtubules. Both a- and b-tubulins are members of the tubulin/FtsZ/CetZ superfamily of proteins, whose eukaryotic and prokaryotic members share the ability to bind guanine nucleotides and participate in the formation of linear polymers. Of all the members of this family only a- and b-tubulins form a dimer that is stable in dilute solutions. Other filament-forming members of this family, such as btubA/B and FtsZ, can form weak dimers but these species are not populated in dilute solutions. The association of the a- and b-tubulin monomers to form the heterodimer involves protein-protein interaction surfaces that are significantly different from those that form the dimer-dimer contact in protofilaments, so that heterodimers are stable in non-polymerizing conditions. We compared the properties of the intra-dimer interface (monomer-monomer) with the properties of the inter-dimer interface (dimer-dimer) in published models of tubulin protofilaments, including curved and straight filaments. Based on these analyzes we predict at least 10-fold enhanced stability of the monomer-monomer association over dimer-dimer association. We tested this prediction by measuring the dimer dissociation constant (Kd) and the critical concentration for polymerization (CC). We also measured the effects of changing the solution composition on dimer Kd (reflecting monomer-monomer association), and on CC (reflecting dimer-dimer association). We examined 4 changes to the solution: addition of ligands 1) GTP or GDP at 0.5 mM, addition of co-solvents 2) urea or 3) Trimethylamine N-oxide (TMAO), each at 0.3 M, and addition of 4) NaCl 0.5 M. Our experimental results demonstrate the enhanced stability and lower sensitivity to solution composition changes of tubulin monomer-monomer association compared to dimer-dimer association.

Control by Potassium of the Size Distribution of Escherichia coli FtsZ Polymers Is Independent of GTPase Activity

The influence of potassium content (at neutral pH and millimolar Mg(2+)) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5'-(α,β-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring.

Importance of a Lys113–Glu195 Intermonomer Ionic Bond in F-actin Stabilization and Regulation by Yeast Formins Bni1p and Bnr1p

Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys(113) of one monomer and Glu(195) of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.

Purification and Characterization of Escherichia coli MreB Protein

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.

Highly Controllable Ring–Chain Equilibrium in Quadruply Hydrogen Bonded Supramolecular Polymers

Electron-rich dioxynaphthalene (DNP) group bridged bifunctional ureidopyrimidinone (UPy) derivatives (L1, L2, and L3) were synthesized, which could form small cyclic monomers, oligomers, or linear supramolecular polymers at certain concentration in solution, to achieve a highly controllable ring–chain equilibrium self-assembling supramolecular system. The ring–chain equilibrium of these supramolecular monomers constructed by different lengths of oligo(ethylene oxide) (oligoEO) chain as spacers were investigated by a combination of techniques, such as 1H NMR, DOSY, single-crystal X-ray diffraction, and viscometry. The experiment results demonstrated that there exists intramolecular π–π stacking interaction between DNP group and intramolecularly dimerized UPy motif in the monomeric cyclic form of these supramolecular monomers, and the strength of this π–π stacking interaction directly depends on the length of the oligoEO chain. Furthermore, strong intramolecular π–π stacking interaction was found to promote self-assembly favorable for intramolecularly cyclic monomerization, leading to a great increase of critical polymerization concentration (CPC). Monomer L1a with the shortest length of oligoEO chain is present as an exclusive type of intramolecularly hydrogen-bonded assembly, namely the cyclic monomers, over a broad concentration range (1.6–500 mM) in solution. Single crystal structure of the cyclic monomer L1b, which is an analogue of L1a, was thoroughly studied. The CPC values of monomer L2 and L3 with longer oligoEO chain are ca. 70 and 23 mM, respectively. However, L2 and L3 could perform selective cyclization over the entire concentration range in solution after threading into the tetracationic cyclobis(paraquat-p-phenylene)cyclophane (CBPQT4+) driven by host–guest interaction, which provides another supramolecular strategy to control ring–chain equilibrium. The combined results may provide new insights into the ring–chain equilibrium and offer valuable information on the understanding of the correlation between supramolecular assistance and polymerizability.

Nuclear actin and myosins at a glance

Actin and myosin II form the archetypical molecular motor complex. Myosin II, like all members of the myosin superfamily, is an actin-activated ATPase that uses the energy released when ATP is hydrolyzed to do work. Although we typically associate work with muscle contraction, cell motility and cell

Studies on the Dissociation and Urea-Induced Unfolding of FtsZ Support the Dimer Nucleus Polymerization Mechanism

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (Kd = 9 μM) indicates a significant fraction (∼10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization.

Thermal stability of carp actin in its polymerized form

We investigated the thermal denaturation of carp F-actin by measuring the loss of polymerization ability. The thermal denaturation rates of carp F-actin were at least 10-fold higher than those of chicken F-actin. Binding of tropomyosin thermally stabilized carp F-actin with no appreciable change in activation energy, suggesting the instability was caused by a high frequency of fragmentation of the actin filaments. A comparison of the critical concentration for polymerization suggested that the subunit–subunit interactions of carp F-actin were indeed weaker than those of chicken F-actin. Furthermore, using fluorescence quenching we showed that the nucleotide binding region of carp F-actin was in a more open conformation. Together, our results suggest that the instability of carp F-actin is a function of both the rate of fragmentation and the dissociation of bound nucleotides.

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary "preference" for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations.

Organization of F-Actin by Avian Smooth Muscle Synaptopodin 2 (Fesselin)

Fesselin is a member of the synaptopodin family of actin binding proteins and it is rich in smooth muscle tissue. Fesselin accelerates actin polymerization in a calcium-calmodulin dependent fashion and forms insoluble aggregates with actin. The polymerization of actin occurs even in the absence of salts usually required to initiate polymerization. The question is whether the properties of fesselin are indicative of a function in forming structures rich in fesselin such as dense bodies and Z-lines. Electron microscopy of actin filaments shows that fesselin initially produces long actin filaments that become organized into thick bundles. The structure of the bundles depends on the ratio of fesselin to actin. Most frequently there are dense parallel ordered bundles. In some cases, the bundles are frayed and thus display single actin filaments. The filaments within a bundle are in-register and have either common initiation or termination points. Decoration with S1 showed that the filaments of a bundle had the same orientation. Actin filaments were occasionally seen to be radiating from densely stained complexes. Solution studies support the idea that fesselin stimulates pointed end growth of actin filaments. That is, fesselin (a) increased the critical concentration for polymerization and (b) induced actin growth even in the presence of barbed end blockers. When added to actin in the absence of other factors, fesselin can nucleate networks of linear actin filaments in which growth occurs primarily at the pointed ends of actin filaments.

α- and β-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening

Internal fragments of alpha- and beta-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5'- and 3'-rapid amplification of cDNA ends. Phytophthora capsici alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC(50)) = 468 +/- 20 microM. Approximately 18.66 +/- 0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 +/- 0.12 residues were accessible in the presence of 200 microM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.