Combinations Of Side Chains Research Articles

Stretchable diketopyrrolopyrrole-based conjugated polymers with asymmetric sidechain designs for field-effect transistor applications

BackgroundThe asymmetric sidechain design has been recently proposed as a potential strategy to improve the mobility-stretchability property of conjugated polymers. In this study, the influences of the asymmetric sidechain designs on the mobility-stretchability properties of DPP-based conjugated polymers are investigated. Three asymmetric sidechain combinations based on a linear alkyl chain (C12), a carbosilane chain (Si), and a siloxane-terminated chain (SiO) are designed to synthesize three polymers, P(SiO-C12), P(Si-C12), and P(SiO-Si). MethodsAll polymers are synthesized via Stille coupling in a microwave reactor. Their basic properties including size exclusion chromatography (SEC), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) are analyzed. Their optical and electrochemical properties are characterized using UV-vis absorption and cyclic voltammetry (CV). The morphology of the polymer films is analyzed through optical microscopy (OM), atomic force microscopy (AFM), and grazing-incidence X-ray diffraction (GIXD). Lastly, the transistor characteristics are measured using a Keithley 4200-SCS semiconductor parameter analyzer (Keithley Instruments Inc.) inside a N2-filled glove box. Significant findingsThe asymmetric SiO/Si sidechain combination enables P(SiO-Si) possess the highest degree of bimodal packing orientation because their branched geometries impose a large steric hindrance to disrupt the chain packing. As benefitting from such bimodal packing orientation, P(SiO-Si) is shown to deliver the highest hole mobility value and the best mobility-stretchability property among these three polymers. It can persist 56.7% of the initial μh value after 800 cycles of 60% strain while P(SiO-C12) and P(Si-C12) can only maintain 2.4% and 4.1% of the initial value. Our results clearly highlight the advantage of the asymmetric SiO/Si sidechain combination on improving the mobility-stretchability property of the derived polymer.

Promiscuity mapping of the S100 protein family using a high-throughput holdup assay

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein–protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.

Reducing the side-chain influences of isoindigo-based polymer donors by backbone fluorination in photovoltaic applications

The effect of synergistic structural engineering (asymmetric side chains and backbone fluorination) for polymer donors in photovoltaic applications is investigated in this study. Six isoindigo (IID)-based polymer donors with slightly different side-chain and backbone designs are composed of a bithiophene (BT) or fluorinated bithiophene moiety to form two series of polymers [PII2T (P1–P3) and PII2TF (P4–P6)] while symmetric/asymmetric side-chain combinations are composed of decyltetradecane branched alkyl side chain (DT) and siloxane-terminated side chain (SiO–C8). The results indicate the different side chain design and backbone fluorination modulate the energy levels, crystalline properties, and molecular packing behaviors of the polymers. P2 and P5 with asymmetric side chains are showed to deliver the highest power conversion efficiency (PCE) in the respective series of PII2T and PII2TF. The morphological analyses reveals that the mismatched side-chain geometry results in a face-on dominated packing to yield superior device performance. Meanwhile, the fluorinated polymers (P4–P6) show superior performance than the corresponding non-fluorinated polymers (P1–P3). Moreover, backbone fluorination largely reduces the performance difference between the polymers with symmetric side chains and asymmetric sides chains. This work demonstrates that the synergistic effect of asymmetric side chains and backbone fluorination can be a rational design towards high-performance conjugated polymers.

Synthesis of helical branched carbodiimide polymers with liquid crystalline properties

ABSTRACT Carbodiimide polymers are synthetic helical polymers that display unique properties depending on the functional groups attached to the polymer backbone. The amidine groups of the backbone present alternating amine- and imine-like groups. The properties of the polycarbodiimides have been studied by varying the sidechains of the polymer from linear to branched. In previous work, different arenes and straight aliphatic chains substituted polycarbodiimides have been used to evaluate the thermal and liquid crystalline properties of these polymers. In this study, we introduced bulky branched aliphatic and octadecyl side chains to the polymer backbone to generate a branched homopolymer and different branched/linear-chain copolymers. The effect of these side-chain combinations on the solid and solution properties of these polycarbodiimides was studied. It was observed that polycarbodiimides containing octadecyl and branched bulky alkyl groups showed different thermal events in the DSC plot at ~30°C. Also, we observed a decrease in thermal stability in the TGA thermogram as we increase the branched alkyl side chains of the polymers. The lyotropic liquid crystallinity was observed in all the branched copolymers that consist of both branched alkyl side chains and octadecyl functional groups in different ratios.

Asymmetric Side-Chain Engineering of Isoindigo-Based Polymers for Improved Stretchability and Applications in Field-Effect Transistors.

Thus far, there is still no study systematically investigating the influence of asymmetric side-chain design on a polymer's stretchability and its associated stretchable device applications. Herein, three kinds of asymmetric side chains consisting of carbosilane side chain (Si-C8), siloxane-terminated side chain (SiO-C8), and decyltetradecane side chain (DT) are engineered in isoindigo-bithiophene (PII2T, P1-P3) and isoindigo-difluorobithiophene (PII2TF, P4-P6) conjugated polymers, and their structure-stretchability correlation is explored in field-effect transistor characterization. It is revealed that owing to the geometric difference between the side chains, different asymmetric side-chain combinations impose distinct influences on the molecular stacking and orientation of the derived polymers. Surprisingly, the combination of asymmetric side chains and backbone fluorination is shown to deliver the best stretchability and mechanical durability of the derived polymer. Consequently, P6 consisting of asymmetric Si-C8/DT side chains and fluorinated backbone possesses the best mobility preservation of 81% at 100% strain with the stretching force perpendicular to the charge-transporting direction. Moreover, it presents 90% mobility retention after 400 stretching-releasing cycles with 60% strain, greatly exceeding the value (36%) of the non-fluorinated counterpart (P3). Our results suggest that the rational design of asymmetric side chains and backbone fluorination provides an efficient way to enhance the intrinsic stretchability of conjugated polymers.

Charge-Transfer Knowledge Graph among Amino Acids Derived from High-Throughput Electronic Structure Calculations for Protein Database.

The charge-transfer coupling is an important component in tight-binding methods. Because of the highly complex chemical structure of biomolecules, the anisotropic feature of charge-transfer couplings in realistic proteins cannot be ignored. In this work, we have performed the first large-scale quantitative assessment of charge-transfer preference by calculating the charge-transfer couplings in all 20 × 20 possible amino acid side-chain combinations, which are extracted from available high-quality structures of thousands of protein complexes. The charge-transfer database quantitatively shows distinct features of charge-transfer couplings among millions of amino acid side-chain combinations. The overall distribution of charge-transfer couplings reveals that only one average or representative structure cannot be regarded as the typical charge-transfer preference in realistic proteins. This work provides us an alternative route to comprehensively understand the charge-transfer couplings for the overall distribution of realistic proteins in the foreseen big data scenario.

Understanding intrinsic plasticizer in vegetable oil-based polyurethane elastomer as enhanced biomaterial

Renewable polyol is of increasing interest as a building block in biomedical elastomer for bearing biodegradable ester group and immaculate functionality. Derived from non-edible vegetable oil, a new class of elastomer was successfully functionalized with MDI and TDI. Crosslink densities were varied by regulating ratio of hydroxyl to diisocyanate (r) at 1/1.0, 1/1.1, and 1/1.2. Produced elastomers were examined by crosslink density, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical analysis, tensile testing, and scanning electron microscopy. The obtained elastomers had subambient glass transition temperature (T g) suggested majority soft segment that acted as a continuous phase with intermediate phase separation. Medium conversion at gel point had enhanced physical properties. Highly elastic mechanical behavior was afforded from combination of side chains and high molecular weight polyol. At r = 1/1.2, MDI-based elastomer showed twofold improvement in Young modulus at slight expense of elongation. TDI-based elastomer accomplished elongation beyond 162%. Branching allophanate and biuret resisted early thermal breakdown by elevating activation energy. Frequency response and kinetic of thermal degradation provided beneficial perspective for elastomer characterization. The vegetable oil-based polyurethane was found able to resemble most of the physical properties of polycaprolactone (PCL)-derived polyurethane.

Improved specificity of serum phosphatidylcholine detection based on side-chain losses during negative electrospray ionization tandem mass spectrometry.

Many current tandem mass spectrometry (MS) methods for measuring phosphatidylcholines (PtdChos) rely only on precursor ion scanning of the common 184 m/z phosphocholine fragment with positive electrospray ionization (+ESI), and thus measure pools of PtdChos rather than specific isoforms. In this paper, we developed and compared an isotope dilution, tandem MS method capable of quantifying PtdChos based on specific fatty acid side-chains to the traditional 184 m/z method. The method is based on the detection of PtdCho ammonium formate (AmF) adduct as parent ions and fatty acid fragment daughter ions under negative electrospray ionization (-ESI). Accuracy, imprecision, and recovery were below 15%, with acceptable linearity (R 2 > 0.99) up to 5μg/mL. We used the method to analyze the distributions of PtdChos with common side-chain combinations among 60 subjects and showed that it was possible for two individuals to have the same PtdCho pool concentration based on detection of the 184 m/z fragment, but up to a fourfold difference in the levels of specific isoforms comprising the pool based on our method. We then compared the results of both methods across 572 patients with mild cognitive impairment (MCI), Alzheimer's disease (AD), or no impairment (NI), which showed that statistically significant associations between specific PtdCho isoforms and AD were masked with the 184 m/z method. Our findings demonstrate the importance of isoform specificity for quantifying PtdChos, and suggest caution when interpreting analytical data based on pools of biomarkers.

Sequence-Specific Recognition of DNA by Proteins: Binding Motifs Discovered Using a Novel Statistical/Computational Analysis.

Decades of intensive experimental studies of the recognition of DNA sequences by proteins have provided us with a view of a diverse and complicated world in which few to no features are shared between individual DNA-binding protein families. The originally conceived direct readout of DNA residue sequences by amino acid side chains offers very limited capacity for sequence recognition, while the effects of the dynamic properties of the interacting partners remain difficult to quantify and almost impossible to generalise. In this work we investigated the energetic characteristics of all DNA residue—amino acid side chain combinations in the conformations found at the interaction interface in a very large set of protein—DNA complexes by the means of empirical potential-based calculations. General specificity-defining criteria were derived and utilised to look beyond the binding motifs considered in previous studies. Linking energetic favourability to the observed geometrical preferences, our approach reveals several additional amino acid motifs which can distinguish between individual DNA bases. Our results remained valid in environments with various dielectric properties.

Effect of Side Chains on Molecular Conformation of Anthracene-Ethynylene-Phenylene-Vinylene Oligomers: A Comparative Density Functional Study With and Without Dispersion Interaction

Using density functional calculations with and without dispersion interaction, we studied the effects of linear octyl and branched 2-ethylhexyl side chains on the oligomer conformation of the conjugated copolymer poly(p-anthracene-ethynylene)-alt-poly(p-phenylene-vinylene). With dispersion included, the branched side chains can cause significant bending of the oligomer backbone, while without dispersion they induce mainly torsional disorder. The oligomers with mainly linear side chains keep good planarity when optimized with and without dispersion. Despite their dramatically different conformations, the calculated absorption spectra of the oligomers with various side chain combinations are very similar, indicating that the conformation of the copolymer is not the main reason for the experimentally observed different spectra of ordered and disordered phases.

1,2,3‐Triazole–Diketopyrrolopyrrole Derivatives with Tunable Solubility and Intermolecular Interactions

Abstract1,2,3‐Triazole rings bearing hydrophobic aliphatic or hydrophilic oligoether chains were easily introduced at the two ends of the conjugated skeleton of bisthiophene–diketopyrrolopyrrole (TDPP) derivatives by simple click cycloaddition reactions. The combination of side chains with different structures and polarities on the triazole rings with the side chains on the N‐atoms of the lactam groups of the TDPP moiety enabled the solubility and the solid‐state spectroscopic properties of the resulting conjugated molecules to be tuned. Formation of nanostructured aggregates and dependence of their spectroscopic behavior on the substitution pattern were investigated.

Current challenges in peptide-based drug discovery.

The central event of each signaling step in biology is biomolecular recognition. Notwithstanding the importance of nucleic acids, carbohydrates, or lipids in ligand-target interactions, the effectors of most signal transduction processes are peptides. These can be fragments of proteins or stand-alone hormones, cytokines, toxins, antimicrobials, and many other types of peptides. At this point there is no good reason to classify peptides by the number of amino acid residues. We consider peptides as any polyamide (or even biopolymer with ester, thioester, or otherwise modified backbone) that can be made on a contemporary chemical peptide synthesizer. The limit in size is greater than the arbitrary cutoff of 50 amino acids set up by the US Food and Drug Administration (Carton and Strohl, 2013) for proteins and far exceeds that of biological recognition elements. While target recognition can occur with as low as a few residues (Ertl et al., 1991), even wide binding groves can be bound by 30–40 residue long peptides. Thus, in principle synthetic peptides can be used to regulate almost all receptor responses. The high specificity and low toxicity of peptide drugs derive from their extremely tight binding to their targets. This is due to the large chemical space the side-chain variations of native amino acids cover. Current databases estimate the total number of valid protein-ligand binding sites at 7700 (Khazanov and Carlson, 2013). Calculation based on 17 variable residues (Cys, Met, and Trp are significantly underrepresented in known ligands), show that an 83,000-member tetrapeptide library can be prepared that will essentially cover all unique protein binding regions. As the median length of an active site is 11 amino-acid residues (Khazanov and Carlson, 2013), designed ligands should also be longer. While historically six-residue positional scanning could identify ligands of receptors or epitopes of monoclonal antibodies (Dooley and Houghten, 1993), in our experience receptor agonists are 9–12 residue long (Otvos et al., 2008, 2011a) much like major histocompatibility complex binding peptides (Appella et al., 1995). Antagonists acting on the same receptor binding sites are somewhat shorter (vide infra). If it is assumed that conformational preferences improve the binding kinetics but only rarely thermodynamics, then the tremendous specificity of side-chain combinations of peptides over six residues in length can be even further expanded by using non-natural residues. Hundreds of appropriately protected and activated non-natural amino acid derivatives, ready for incorporation into synthetic peptides, are commercially available and indeed are frequently explored in peptidebased drug design. Importantly, chemical biology has provided both backbone and side-chain combinations for exploring an enormous chemical space and is expected to supply peptide chemists with further building blocks suitable for identifying close-to-ideal agonists and antagonists of any biologically important target. The selectivity of peptide drugs for their target is highlighted by the elevated success rate in clinical trials. According to a biotechnology report (Thomas, 2013), of the 40 approved drugs in 2012, five (12.5%) were peptides compared to 28 small molecule drugs and two monoclonal antibodies (in addition to three enzymes, a cell-based drug and a vaccine). However, in a recent report, the total number of peptide approvals between 2001 and 2012 was 19 (Kaspar and Reichert, 2013). Due to the low number of drug approvals, any particularly successful year can bias the ratios significantly. According to another report, the overall success rate of all drugs entering clinical trials is just 10.4% (Hay et al., 2014). Sixty-five percent of small molecules proceed from Phase I to Phase II in non-oncology applications, a figure identical for peptide/protein drugs. Interestingly peptides/proteins outperform small molecules at the Phase II → Phase III transition stage with 29% for small molecules and 42% for the larger drug candidates. While peptides have traditionally been considered safe in Phase I clinical trials, the public perception is that they are less beneficial in late clinical trials when they are compared side-by-side with different types of treatment modalities. It must be mentioned that peptides are less successful in oncology than in other applications. The cost of large scale peptide production might well-exceed those of small molecule drugs, but if one considers the total cost of the drug development process, the active pharmaceutical ingredient expense will remain under 3% (Otvos, 2014a). In direct opposition to concerns with expensive peptides, the increased clinical success rate, and thus, overall expense/approved drug ratio compared to small molecule chemical entities, make peptide drug development particularly attractive. The biochemical processes that activated receptors directly or indirectly regulate include protein phosphorylation, nucleic acid transcription, ion transport, and a series of enzyme activities (Yan

Thiazolopyridine Ureas as Novel Antitubercular Agents Acting through Inhibition of DNA Gyrase B

A pharmacophore-based search led to the identification of thiazolopyridine ureas as a novel scaffold with antitubercular activity acting through inhibition of DNA Gyrase B (GyrB) ATPase. Evaluation of the binding mode of thiazolopyridines in a Mycobacterium tuberculosis (Mtb) GyrB homology model prompted exploration of the side chains at the thiazolopyridine ring C-5 position to access the ribose/solvent pocket. Potent compounds with GyrB IC50 ≤ 1 nM and Mtb MIC ≤ 0.1 μM were obtained with certain combinations of side chains at the C-5 position and heterocycles at the C-6 position of the thiazolopyridine core. Substitutions at C-5 also enabled optimization of the physicochemical properties. Representative compounds were cocrystallized with Streptococcus pneumoniae (Spn) ParE; these confirmed the binding modes predicted by the homology model. The target link to GyrB was confirmed by genetic mapping of the mutations conferring resistance to thiazolopyridine ureas. The compounds are bactericidal in vitro and efficacious in vivo in an acute murine model of tuberculosis.

Disentangling the impact of side chains and fluorine substituents of conjugated donor polymers on the performance of photovoltaic blends

Side chains and fluorine (F) substituents on conjugated polymers have shown significant impact on the photovoltaic properties of polymer-based bulk heterojunction (BHJ) solar cells, but their respective impact is largely studied independently. In order to disentangle the effect of side chains and F substituents, we comprehensively investigate a series of conjugated polymers with an identical backbone (PNDT–DTBT) but different combinations of side chains and F substituents. Surprisingly, these seemingly marginal changes to the polymer backbone strongly influence the morphology and structure in BHJ thin films (e.g., domain size/purity and the relative orientation of polymer crystallites), as manifested by resonant soft X-ray scattering (R-SoXS) and grazing-incidence wide-angle X-ray scattering (GI-WAXS), thereby exerting significant impact on the photovoltaic properties of these conjugated polymer-based BHJ cells. Devices based on the polymer with long bulky side chains and F substituents (C8,4-C6,2F) simultaneously exhibit large open circuit voltage (Voc), high short circuit current (Jsc) and good fill factor (FF), with an efficiency as high as 5.6% for this series of PNDT–DTBT polymers.

Proton Conduction in Exchange Membranes across Multiple Length Scales

Concerns over global climate change associated with fossil-fuel consumption continue to drive the development of electrochemical alternatives for energy technology. Proton exchange fuel cells are a particularly promising technology for stationary power generation, mobile electronics, and hybrid engines in automobiles. For these devices to work efficiently, direct electrical contacts between the anode and cathode must be avoided; hence, the separator material must be electronically insulating but highly proton conductive. As a result, researchers have examined a variety of polymer electrolyte materials for use as membranes in these systems. In the optimization of the membrane, researchers are seeking high proton conductivity, low electronic conduction, and mechanical stability with the inclusion of water in the polymer matrix. A considerable number of potential polymer backbone and side chain combinations have been synthesized to meet these requirements, and computational studies can assist in the challenge of designing the next generation of technologically relevant membranes. Such studies can also be integrated in a feedback loop with experiment to improve fuel cell performance. However, to accurately simulate the currently favored class of membranes, perfluorosulfonic acid containing moieties, several difficulties must be addressed including a proper treatment of the proton-hopping mechanism through the membrane and the formation of nanophase-separated water networks. We discuss our recent efforts to address these difficulties using methods that push the limits of computer simulation and expand on previous theoretical developments. We describe recent advances in the multistate empirical valence bond (MS-EVB) method that can probe proton diffusion at the nanometer-length scale and accurately model the so-called Grotthuss shuttling mechanism for proton diffusion in water. Using both classical molecular dynamics and coarse-grained descriptions that replace atomistic representations with collective coordinates, we investigated the proton conductivity of polymer membrane structure as a function of hydration level. Nanometer-sized water channels form torturous pathways that are traversed by the charges during fuel cell operation. Using a combination of coarse-grained membrane structure and novel multiscale methods, we demonstrate emerging approaches to treat proton motion at the mesoscale in these complex materials.

Design, Synthesis, and Validation of a β-Turn Mimetic Library Targeting Protein–Protein and Peptide–Receptor Interactions

The design and synthesis of a β-turn mimetic library as a key component of a small-molecule library targeting the major recognition motifs involved in protein-protein interactions is described. Analysis of a geometric characterization of 10,245 β-turns in the protein data bank (PDB) suggested that trans-pyrrolidine-3,4-dicarboxamide could serve as an effective and synthetically accessible library template. This was confirmed by initially screening select compounds against a series of peptide-activated GPCRs that recognize a β-turn structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small molecules with high affinities (K(i) = 390 and 23 nM, respectively) for the κ-opioid receptor (KOR). Consistent with the screening capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20 compounds, providing a total of 4200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Unique to the design and because of the C(2) symmetry of the template, a typical 20 × 20 × 20-mix (8000 compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid residues reduces to a 210 × 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared using a solution-phase synthetic protocol with liquid-liquid or liquid-solid extractions for purification and conducted on a scale that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR, MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands but also additional side-chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as K(i) = 80 nM for KOR). A key insight to emerge from the studies is that the phenol of Tyr in endogenous ligands bearing the H-Tyr-Pro-Trp/Phe-Phe-NH(2) β-turn is important for MOR binding but may not be important for KOR (accommodated, but not preferred) and that the resulting selectivity for KOR observed with its removal can be increased by replacing the phenol OH with a chlorine substituent, further enhancing KOR affinity.

Sub-cellular internalization and organ specific oral elivery of PABA nanoparticles by side chain variation

BackgroundOrganic nanomaterials having specific biological properties play important roles in in vivo delivery and clearance from the live cells. To develop orally deliverable nanomaterials for different biological applications, we have synthesized several fluorescently labelled, self-assembled PABA nanoparticles using possible acid side chain combinations and tested against insect and human cell lines and in vivo animal model. Flurophores attached to nanostructures help in rapid in vivo screening and tracking through complex tissues. The sub-cellular internalization mechanism of the conjugates was determined. A set of physio-chemical parameters of engineered nanoskeletons were also defined that is critical for preferred uptake in multiple organs of live Drosophila.ResultsThe variability of side chains alter size, shape and surface texture of each nanomaterial that lead to differential uptake in human and insect cells and to different internal organs in live Drosophila via energy dependent endocytosis. Our results showed that physical and chemical properties of C-11 and C-16 acid chain are best fitted for delivery to complex organs in Drosophila. However a distinct difference in uptake of same nanoparticle in human and insect cells postulated that different host cell physiology plays a critical role in the uptake mechanism.ConclusionsThe physical and chemical properties of the nanoparticle produced by variation in the acid side chains that modify size and shape of engineered nanostructure and their interplay with host cell physiology might be the major criteria for their differential uptake to different internal organs.

Design, Production and Molecular Structure of a New Family of Artificial Alpha-helicoidal Repeat Proteins (αRep) Based on Thermostable HEAT-like Repeats

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable ( T m > 70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners.

Precise structural analysis of α-helical polypeptide by quantum-chemical calculation related to reciprocal side-chain combination of two L-phenylalanine residues

To clarify the positive role of side-chain conformation in the stability of protein secondary structure (main-chain conformation), we successfully calculated the optimization structure of a series of well-defined α-helical octadecapeptides composed of two L-phenylalanine (Phe) and 16 L-alanine (Ala) residues, based on the molecular orbital calculation with density functional theory (DFT/B3LYP/6-31G(d)). From the total energy calculation and the precise secondary structural analysis, we found that the conformational stability of the α-helix is closely related to the reciprocal side-chain combinations (such as positional relation and side-chain conformation) of two Phe residues in this system. Furthermore, we demonstrated that the 1H, 13C, 15N and 17O isotropic chemical shifts of each Phe residue depend on the respective side-chain conformations of the Phe residue.

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO(2) fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg(2+) for catalysis and, like RuBisCO, was stimulated by CO(2). Four residues that are essential for the enolization reaction of RuBisCO, Lys(175), Lys(201), Asp(203), and Glu(204), were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys(123), the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K(i) value of 103 mum. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.