Symmetry‐Directed Self‐Assembly of a Tetrahedral Protein Cage Mediated by de Novo‐Designed Coiled Coils

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of protein–protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiled‐coil domains to assemble proteins into higher‐order structures. Here, we demonstrate the assembly of a trimeric enzyme into a well‐defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiled‐coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiled‐coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.     

An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution

Recent studies in the field of de novo protein design have focused on the construction of native-like structures. Here we describe the design and characterization of an isoleucine zipper peptide intended to form a parallel triple-stranded coiled coil. To obtain the native-like structural uniqueness, the hydrophobic interface of the peptide consists of beta-branched Ile residues for complementary side chain packing. The peptide forms a stable triple-stranded coiled coil, as determined by circular dichroism and sedimentation equilibrium analyses. A fluorescence quenching assay after the incorporation of acridine revealed a parallel orientation of the peptides. The structural uniqueness of the coiled coil was confirmed by proton- deuterium amide hydrogen exchange and hydrophobic dye binding. The peptide contains amide protons with hydrogen exchange rates that are approximately an order of magnitude slower than those expected if the exchange occurred via global unfolding. A hydrophobic dye does not bind to the peptide. These results strongly suggest that the peptide folds into a well-packed structure that is very similar to the native state of a natural protein. Thus, Ile residues in the hydrophobic interface can improve the side chain packing, which can impart native-like structural uniqueness to the designed coiled coil.      

A new expression system for protein crystallization using trimeric coiled-coil adaptors

We repeatedly experienced difficulties in obtaining pure protein of a defined oligomeric state when expressing domains that consist partially or entirely of coiled coils. We therefore modified an established expression vector, pASK-IBA, to generate N- and C-terminal fusions of the cloned domain in heptad register with the GCN4 leucine zipper. GCN4 is a well-characterized coiled coil, for which stable dimeric, trimeric and tetrameric forms exist. To test this expression system, we produced a series of constructs derived from the trimeric autotransporter adhesin STM3691 of Salmonella (SadA), which has a highly repetitive structure punctuated by coiled-coil regions. The constructs begin and end with predicted coiled-coil segments of SadA, each fused in the correct heptad register to the trimeric form of GCN4, GCN4pII. All constructs were expressed at high levels, trimerized either natively or after refolding from inclusion bodies, and yielded crystals that diffracted to high resolution. Thus, fusion to GCN4pII allows for the efficient expression and crystallization of proteins containing trimeric coiled coils. The structure of short constructs can be solved conveniently by molecular replacement using the known GCN4 structure as a search model. The system can be adapted for constructs with dimeric or tetrameric coiled coils, using the corresponding GCN4 variants.     

Optimizing the Photocontrol of bZIP Coiled Coils with Azobenzene Crosslinkers: Role of the Crosslinking Site

DNA binding by bZIP‐type coiled‐coil proteins can be inhibited by dominant negative versions of the proteins in which the N‐terminal basic region is replaced by an acidic extension. Photocontrol of bZIP function can be achieved by introducing intramolecular azobenzene‐based crosslinkers into dominant negatives. We show that the largest degree of photocontrol is achieved when the crosslinker is introduced into the zipper region of the dominant negative between Cys residues placed at f sites in the heptad segment showing the highest intrinsic helical propensity. The overall affinity of the dominant negative can then be tuned by varying the length of the acidic extension.     

Segregation of resin‐bound peptides during solid‐phase synthesis

To compare the segregation ability of 1,4‐butanediol dimethacrylate‐crosslinked polystyrene (BDDMA‐PS) and divinylbenzene‐crosslinked polystyrene (DVB‐PS), a set of difficult sequence peptides characterized by high‐arithmetic‐average β‐sheet stabilizing potential (SP_β) and low‐stepwise arithmetic average random coil conformational parameter (P_c*) were synthesized on both supports (～ 2 mmol Cl g^?1) under identical conditions. The yield and purity of the peptides obtained from BDDMS‐PS resin were higher than from DVB‐PS resin. The synthetic efficiency of the new support was found to be its ability to suppress the aggregation of growing peptide chains by β‐sheet formation. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1717–1723, 2002     

Predicting leucine zipper structures from sequence

The leucine zipper structure is adopted by one family of thecoiled coil proteins. Leucine zippers have a characteristicleucine repeat: Leu–X₆–Leu–X₆–Leu–X₆–Leu(where X may be any residue). However, many sequences have theleucine repeat, but do not adopt the leucine zipper structure(we shall refer to these as non-zippers). We have found andanalyzed residue pair patterns that allow one to identify correctly90% of leucine zippers and 97% of non-zippers. Simpler analyses,based on the frequency of occurrence of residues at certainpositions, specify, at most, 65% of zippers and 80–90%of non-zippers. Both short and long patterns contribute to thesuccessful discrimination of leucine zippers from non-zippers.A number of these patterns involve hydrophobic residues thatwould be placed on the solvent-exposed surface of the helix,were the sequence to adopt a leucine zipper structure. Thus,an analysis of protein sequences has allowed us to improve discriminationbetween leucine zippers and non-zippers, and has provided somefurther insight into the physical factors influencing the leucinezipper structure.     

Versatile Interacting Peptide (VIP) Tags for Labeling Proteins with Bright Chemical Reporters

Fluorescence microscopy is an essential tool for the biosciences, enabling the direct observation of proteins in their cellular environment. New methods that facilitate attachment of photostable synthetic fluorophores with genetic specificity are needed to advance the frontiers of biological imaging. Here, we describe a new set of small, selective, genetically encoded tags for proteins based on a heterodimeric coiled‐coil interaction between two peptides: CoilY and CoilZ. Proteins expressed as a fusion to CoilZ were selectively labeled with the complementary CoilY fluorescent probe peptide. Fluorophore‐labeled target proteins were readily detected in cell lysates with high specificity and sensitivity. We found that these versatile interacting peptide (VIP) tags allowed rapid and specific delivery of bright organic dyes or quantum dots to proteins displayed on living cells. Additionally, we validated that either CoilY or CoilZ could serve as the VIP tag, which enabled us to observe two distinct cell‐surface protein targets with this one heterodimeric pair.     

Dicer Nuclease‐Promoted Production of Let7a‐1 MicroRNA Is Enhanced in the Presence of Tryptophan‐Containing Amphiphilic Peptides

A library of Trp‐containing amphiphilic peptides was synthesized and screened for the ability to bind to pre‐miRNA targets. Two members of this family, peptides Ac‐WKKLLKWLKKLLKLAG‐NH₂ ( 2 b ) and Ac‐WKKLLKWLKKLLDabLAG‐NH₂ ( 4 b ) were found to have nanomolar binding affinities to pre‐let7a‐1. Peptides 2 b and 4 b caused an increase in the in vitro Dicer cleavage of pre‐let7a‐1. This observation was confirmed by a cell‐based assay, the results of which show an up to 50 % increase in Dicer activity. Enhanced expression of let7a‐1 promoted by the peptides results in specific reductions of target mRNAs. The results of a microarray study show that lower amount of fluctuating genes are generated in the presence of 2 b or 4 b , relative to that from exogenous delivery of let7a‐1. Because peptides 2 b and 4 b promote enhanced formation of mature let7a‐1 only at the endogenous miRNA level, this specifically controls genes related to let7a‐1.     

Mitochondria‐Penetrating Peptides: Sequence Effects and Model Cargo Transport

A class of mitochondria‐penetrating peptides (MPPs) was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were monitored, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of N‐ and C‐terminal cargo were tested to determine if there are limitations to the lipophilicity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. The results reported demonstrate that these peptide sequences are versatile transporters that will have a range of biological applications.     

Anti‐HIV‐1 Peptide Derivatives Based on the HIV‐1 Co‐receptor CXCR4

The human immunodeficiency virus type 1 (HIV‐1) uses CD4 and the co‐receptor CCR5 or CXCR4 in the process of cell entry. The negatively charged extracellular domains of CXCR4 (CXCR4‐ED) interact with positive charges on the V3 loop of gp120, facilitating binding via electrostatic interactions. The presence of highly conserved positively charged residues in the V3 loop suggests that CXCR4‐ED‐derived inhibitors might be broadly effective inhibitors. Synthetic peptide derivatives were evaluated for anti‐HIV‐1 activity. The 39‐mer extracellular N‐terminal region (NT) was divided into three fragments with 10‐mer overlapping sites ( N1 – N3 ), and these linear peptides were synthesized. Peptide N1 contains Met 1–Asp 20 and shows significant anti‐HIV‐1 activity. Extracellular loops 1 and 2 (ECL1 and 2) were mimicked by cyclic peptides C1 and C2 , which were synthesized by chemoselective cyclization. Cyclic peptides C1 and C2 show higher anti‐HIV‐1 activity than their linear peptide counterparts, L1 and L2 . The cytotoxicities of C1 and C2 are lower than those of L1 and L2 . These results indicate that Met 1–Asp 20 segments of the NT and cyclic peptides of ECL1 and ECL2 are potent anti‐HIV‐1 drug candidates.     

Self‐assembly of rod‐coil molecules into lateral chain‐length‐dependent supramolecular organization

In this article, we report synthesis and characterization of the self‐assembly behavior of coil‐ rod‐coil molecules, consisting of four biphenyls and a p‐terphenyl unit linked together with ether bonds as a rod segment. These molecules contain lateral methyl or ethoxymethyl groups at 2 and 5 positions of the middle benzene ring of p‐terphenyl. The self‐assembling behavior of these materials was investigated by means of DSC, POM, and SAXS in the bulk state. The results reveal that self‐assembling behavior of these molecules is dramatically influenced by a lateral methyl or ethoxymethyl groups in the middle of rod segment. In addition, molecule with PEO (DP = 17) coil chains of identical coil volume fraction to the corresponding molecule connected by PPO (DP = 12) coil chains, shows diverse self‐organizing behavior that may result from the parameters of cross‐sectional area of coil segment and the steric hindrance at the rod/coil interface. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

An Injectable Self‐Healing Multifluorescent Hydrogel Formed by Engineered Coiled‐Coil Polypeptide and Quantum Dots

An injectable and self‐healing multifluorescent hydrogel system based on engineered coiled‐coil polypeptide and CdSe@ZnS quantum dots (QDs) is developed. The mechanical properties of the PC₁₀A‐QD hydrogel are able to be tuned by changing the concentrations of PC₁₀A and QDs. The G′ of PC₁₀A hydrogel increases from 800 to 1000 Pa by doping 6 nm oil‐soluble CdSe@ZnS QDs. The PC₁₀A‐QD hydrogel can easily pass through a 26‐gauge needle without clogging. In addition, through interfacial assembly of PC₁₀A polypeptide on the surface of the PC₁₀A‐QD hydrogel, each of these hydrogel can self‐assemble into a multifluorescent hydrogel. This approach for preparation of injectable self‐healing multifluorescent hydrogels is expected to apply in biomedicine.     

Guanidinylation: A simple way to fabricate cell penetrating peptide analogue‐modified chitosan vector for enhanced gene delivery

In view of the analogous transmembrane function to cell penetrating peptides, guanidine group was incorporated into chitosan by chemical modification to enhance the transfection performance of chitosan vectors. Guanidinylated chitosan (GCS) was shown to be well soluble in neutral aqueous solution. The interaction between GCS with plasmid DNA was characterized by agarose retardation experiment and ethidium bromide displacement assay. GCS formed more stable complexes with DNA under physiological pH than chitosan. The transfection efficiency of GCS was evaluated employing COS‐7 cell line—GCS polyplexes demonstrated higher transfection efficiency and lower cytotoxicity relative to chitosan. The optimum efficiency of GCS was achieved in the vicinity of the critical complexing ratio. The results of flow cytometry indicated that guanidinylation promoted an eightfold increase in the cell uptake. The study revealed that guanidinylated chitosan is a promising candidate as an effective nonviral vector for in vivo gene delivery. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Synthesis and characterization of novel poly(amide imide)s based on bis(p‐amidobenzoic acid)‐n‐trimellitylimido‐L‐leucine

N‐Trimellitylimido‐L ‐leucine was reacted with thionyl chloride, and N‐trimellitylimido‐L ‐leucine diacid chloride was obtained in a quantitative yield. The reaction of this diacid chloride with p‐aminobenzoic acid was performed in dry tetrahydrofuran, and bis(p‐amidobenzoic acid)‐N‐trimellitylimido‐L ‐leucine (5) was obtained as a novel optically active aromatic imide–amide diacid monomer in a high yield. The direct polycondensation reaction of the monomer imide–amide diacid 5 with 4,4′‐diaminodiphenylsulfone, 4,4′‐diaminodiphenylether, 1,4‐phenylenediamine, 1,3‐phenylenediamine, 2,4‐diaminotoluene, and benzidine (4,4′‐diaminobiphenyl) was carried out in a medium consisting of triphenyl phosphite, N‐methyl‐2‐pyrolidone, pyridine, and calcium chloride. The resulting novel poly(amide imide)s (PAIs), with inherent viscosities of 0.22–0.52 dL g^?1, were obtained in high yields, were optically active, and had moderate thermal stability. All of the compounds were fully characterized with IR spectroscopy, elemental analyses, and specific rotation. Some structural characterization and physical properties of these new optically active PAIs are reported. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 35–43, 2002; DOI 10.1002/app.10181     

A Helix‐Stabilized Cell‐Penetrating Peptide as an Intracellular Delivery Tool

Two types of cationic cyclic α,α‐disubstituted α‐amino acids: Api (which possesses a lysine mimic side chain) and Api^C2Gu (which possesses an arginine mimic side chain), were developed. These amino acids were incorporated into an arginine‐based peptide sequence [(l ‐Arg‐l ‐Arg‐dAA)₃: dAA=Api or Api^C2Gu], and the relationship between the secondary structures of the resulting peptides and their ability to pass through cell membranes was investigated. The peptide containing Api^C2Gu formed a stable α‐helical structure and was more effective at penetrating cells than the nonhelical Arg nonapeptide (R₉). Furthermore, the peptide was able to deliver plasmid DNA into various types of cells in a highly efficient manner.     

Comparative Performance Analysis of Anode‐Supported Micro‐Tubular SOFCs with Different Current‐Collection Architectures

In this study, the performances of single micro‐tubular solid oxide fuel cells based on the NiO–YSZ/YSZ/LSM system with two different current‐collection architectures were compared. In the first case, a straight Ni wire was inserted within the hole of the cell before the electrochemical testing, and in the second case, a coil integrated‐current collector within the anode layer was already arranged for electrical connections during cell processing. The current produced in each case was collected from double terminal and the performance of the cells was estimated by electrochemical I–V characterization. The maximum power outputs generated in the cells with the integrated‐current collector and the common current‐collection architectures were of ∼200 and ∼55 mW cm^–2, respectively at 800 °C under a wet H₂ fuel flow.