Natural Composite Systems for Bioinspired Materials

Abstract

From a relatively limited selection of base materials, nature has steered the development of truly remarkable materials. The simplest and often overlooked organisms have demonstrated the ability to manufacture multi-faceted, molecular-level hierarchical structures that combine mechanical properties rarely seen in synthetic materials. Indeed, these natural composite systems, composed of an array of intricately arranged and functionally relevant organic and inorganic substances serve as inspiration for materials design. A better understanding of these composite systems, specifically at the interface of the hetero-assemblies, would encourage faster development of environmentally friendly “green” materials with molecular level specificities.

Citation

(10.1007/978-3-319-39196-0_7)
LabLessons: Effects of Electronic Prelabs on Student Engagement and Performance

Abstract

Lab instructors, for both high school and undergraduate college level courses, face issues of constricted time within the lab period and limited student engagement with prelab materials. To address these issues, an online prelab delivery system named LabLessons is developed and tested out in a high school chemistry classroom. The system supplements the laboratory experience by providing visualizations and simulations of concepts to prepare students for the practical experiments. The system requires students to answer prelab questions online, which provides immediate feedback and cuts down on last minute copying of answers that instructors anecdotally reported with paper laboratories. Empirical results demonstrate the effectiveness and improved outcomes for students who have used LabLessons. In addition, the ease of use of the system and better preparedness for the lab is noted by the instructor.

Citation

(10.1021/acs.jchemed.6b00394)
Interactive Online Physics Labs Increase High School Students’ Interest

Abstract

Here we describe the incorporation of a web-based application focusing on circuits for the physics high school classroom as part of an outreach program. The program involves college mentors creating and implementing science lessons in collaboration with the classroom teacher. Focusing on the challenge of understanding circuit design, a technology rich module is employed to improve learning and motivation of the students. The students’ conceptual understanding as well as interest in circuits was increased, the college mentors earned valuable teaching and mentoring experience and the teacher enjoyed more one-on-one time as well as assistance with students.

Citation

(10.3926/jotse.191)
Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials for Small Molecule Delivery

Abstract

We have fabricated protein polymer-gold nanoparticle (P-GNP) nanocomposites that exhibit enhanced binding and delivery properties of the small hydrophobic molecule drug, curcumin, to the model breast cancer cell line, MCF-7. These hybrid biomaterials are constructed via in situ GNP templated-synthesis with genetically engineered histidine tags. The P-GNP nanocomposites exhibit enhanced small molecule loading, sustained release and increased uptake by MCF-7 cells. When compared to the proteins polymers alone, the P-GNPs demonstrated a greater than 7-fold increase in curcumin binding, a nearly 50% slower release profile and more than 2-fold increase in cellular uptake of curcumin. These results suggest that P-GNP nanocomposites serve as promising candidates for drug delivery vehicles

Citation

(10.4172/2157-7439.1000356)
Exploring the potential of engineered coiled-coil protein microfibers in drug delivery

Editorial

Citation

(10.4155/tde.15.19)
Tunable Conformation-Dependent Engineered Protein·Gold Nanoparticle Nanocomposites

Abstract

We demonstrate the fabrication of protein·gold nanoparticle (AuNP) nanocomposites in situ, leading to distinct assemblies dependent upon protein secondary structure. In the presence of pentameric coiled-coil proteins C and Q, which contain histidine tags and have helicities of 54 and 65%, respectively, templation of AuNP results in precipitation of the protein· AuNP composites with AuNPs 6.5 nm in diameter, creating macromolecular assemblies on the micrometer scale. In the absence of the histidine tags, the resulting Cx and Qx proteins, which exhibit lower helicities of 37 and 45%, respectively, stabilize soluble protein·AuNP composites with AuNPs 4.5 nm in diameter for several days without aggregating. By manipulating protein structure via external triggers, such as TFE, we obtain control over the macromolecular conformation and overall physicochemical properties. These hybrid protein·AuNP assemblies can be readily deposited on electrodes, where they can serve as a tunable bionanocomposite kinetic barrier.

Citation

(10.1021/acs.biomac.5b00098)
Influence of Fluorination on Protein Engineered Coiled-coil Fibers

Abstract

We describe the design and characterization of fluorinated coiled-coil proteins able to assemble into robust nanoand microfibers. Fluorination is achieved biosynthetically by residue-specific incorporation of 5,5,5-trifluoroleucine (TFL). The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal denaturation compared to their nonfluorinated counterparts, C and Q. The fluorinated proteins demonstrate enhanced fiber assembly at pH 8.0 with higher order structure in contrast to nonfluorinated proteins, which are unable to form fibers under the same conditions. Ionic strength dependent fiber assembly is observed for fluorinated as well as wild-type proteins in which the fluorinated proteins exhibited more stable, thicker fibers. The fluorinated and nonfluorinated proteins reveal metal ion-dependent small molecule recognition and supramolecular assemblies. In the presence of Zn (II), enhanced thermal stability and fiber assembly is observed for the fluorinated proteins and their nonfluorinated counterparts. Whereas Ni (II) promotes aggregation with no fiber assembly, the stabilization of α-helix by Zn (II) results in enhanced binding to curcumin by the fluorinated proteins. Surprisingly, the nonfluorinated proteins exhibit multiplefold increase in curcumin binding in the presence of Zn (II). In the context of the growing number of protein-based fiber assemblies, these fluorinated coiled-coil proteins introduce a new paradigm in the development of highly stable, robust selfassembling fibers under more physiologically relevant pH conditions that promotes the binding and release of small molecules in response to external cues.

Citation

(10.1021/bm5019062)
Integrating Technology in STEM Education

Abstract

Students have access to the Internet at their fngertps via e-tablets and smart phones. However, the STEM felds are struggling to remain relevant in students’ lives outside the classroom. In an efort to improve high school science curricula and to keep students engaged in the classroom, we developed a technology-rich bioengineering summer program for high school students in grades 9-12. The program utlized touch screen technology in conjuncton with hands-on experiments and traditonal lecturing to create an entertaining, relevant, and efectve classroom experience.

Citation

(10.3926/jotse.124)
Using Touch-Screen Technology, Apps, and Blogs to Engage and Sustain High School Students' Interest in Chemistry Topics

Abstract

As part of an outreach program, we integrated chemistry apps with blogging to enhance the learning experience of students in and outside the classroom. Our outreach program involved college mentors who participated in the development and implementation of chemistry lessons alongside the classroom teacher. Three technology-rich modules that focused on molecules, balancing equations, and nuclear chemistry were taught to high school students. Feedback-oriented and interactive lessons through apps with blogging were used to engage the students with the instructors as well as their peers. The combination of blogging with interactive apps increased student involvement and sustained their interests in the chemistry topics covered by the modules. The students were divided into experimental and control groups. The experimental groups were required to use the blog, where students viewed the questions and uploaded their answers and comments. The control group did not have access to the blog during the modules. Performance, class participation, and interest in STEM fields by the groups were examined. The students received a personalized, interactive learning experience in chemistry, the college mentors gained teaching and mentoring experience, and the teacher received assistance in implementing technology in the classroom.

Citation

(10.1021/ed500234z)
Engineered Coiled-Coil Protein Microfibers

Abstract

The fabrication of de novo proteins able to self-assemble on the nano- to meso-length scales is critical in the development of protein-based biomaterials in nanotechnology and medicine. Here we report the design and characterization of a protein engineered coiled-coil that not only assembles into microfibers, but also can bind hydrophobic small molecules. Under ambient conditions, the protein forms fibers with nanoscale structure possessing large aspect ratios formed by bundles of α-helical homopentameric assemblies, which further assemble into mesoscale fibers in the presence of curcumin through aggregation. Surprisingly, these biosynthesized fibers are able to form in conditions of remarkably low concentrations. Unlike previously designed coiled-coil fibers, these engineered protein microfibers can bind the small molecule curcumin throughout the assembly, serving as a depot for encapsulation and delivery of other chemical agents within protein-based 3D microenvironments.

*highlighted in http://www.fiercedrugdelivery.com/press-releases/nyu-researchers-break-nano-barrier-engineer-first-protein-microfiber

Citation

(10.1021/bm5004948)
Improved Stability and Half-life of Fluorinated Phosphotriesterase using Rosetta

Abstract

Recently we demonstrated that incorporating p-fluorophenylalanine (pFF) into phosphotriesterase dramatically improved folding, thereby leading to enhanced stability and function at elevated temperatures. To further improve the stability of the fluorinated enzyme, Rosetta was used to identify multiple potential stabilizing mutations. One such variant, pFF-F104A, exhibited enhanced activity at elevated temperature and maintained activity over many days in solution at room temperature.

*highlighted in http://www.medicalnewstoday.com/articles/280450.php

Citation

(10.1002/cbic.201402062)
Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex

Abstract

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP·DNA(8:1)·FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat·FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP·DNA(8:1)·FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.

Citation

(10.1016/j.biomaterials.2014.05.005)
Long-term efficient gene delivery using polyethylenimine with modified Tat peptide

Abstract

Polyethylenimine (PEI), a cationic polymer, has been widely studied and shown great promise as an efficient gene delivery vehicle. Likewise, the HIV-1 Tat peptide, a cell-permeable peptide, has been successfully used for intracellular gene delivery. To improve the favorable properties of these two vectors, we combine PEI with the modified Tat peptide sequence bearing histidine and cysteine residues (mTat). In vitro mTat/PEI-mediated transfection was evaluated by luciferase expression plasmid in two cell types. mTat/PEI produced significant improvement (≈5-fold) in transfection efficiency of both cell lines with little cytotoxicity when compared to mTat alone, PEI alone, or four commercial reagents. The particle size of mTat/PEI/DNA complex was significantly smaller than mTat or PEI alone, and it was correlated with higher transfection efficiency. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI transfection. In contrast, chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not. This suggested caveolae-mediated endocytosis as the transfection mechanism. Furthermore, the results of in vivo studies showed that animals administered mTat/PEI/DNA intramuscularly had significantly higher and longer luciferase expression (≈7 months) than those with mTat/DNA, PEI/DNA, or DNA alone, without any associated toxicity. The combination of mTat with PEI could significantly improve transfection efficiency, expanding the potential use as a non-viral gene vector both in vitro and in vivo.

Citation

(10.1016/j.biomaterials.2013.11.012)
Bionanocomposites: Differential Effects of Cellulose Nanocrystals on Protein Diblock Copolymers

Abstract

We investigate the effects of mixing a colloidal suspension of tunicate-derived cellulose nanocrystals (t-CNCs) with aqueous colloidal suspensions of two protein diblock copolymers, EC and CE, which bear two different self-assembling domains (SADs) derived from elastin (E) and the coiled-coil region of cartilage oligomeric matrix protein (C). The resulting aqueous mixtures reveal improved mechanical integrity for the CE+t-CNC mixture, which exhibits an elastic gel network. This is in contrast to EC+t-CNC, which does not form a gel, indicating that block orientation influences the ability to interact with t-CNCs. Surface analysis and interfacial characterization indicate that the differential mechanical properties of the two samples are due to the prevalent display of the E domain by CE, which interacts more with t-CNCs leading to a stronger network with t-CNCs. On the other hand, EC, which is predominantly C-rich on its surface, does not interact as much with t-CNCs. This suggests that the surface characteristics of the protein polymers, due to folding and self-assembly, are important factors for the interactions with t-CNCs, and a significant influence on the overall mechanical properties. These results have interesting implications for the understanding of natural composite biomaterials and the development of artificially assembled bionanocomposites.

Citation

(10.1021/bm401304w)
Increased osseointegration effect of bone morphogenetic protein 2 on dental implants: An in vivo study

Abstract

Application of recombinant human bone morphogenetic protein 2 (rhBMP-2) to implant surfaces has been of great interest due to its osteoinductive potential. However, the optimal coating methodology has not been clarified. The objective of the study was to determine whether the application of rhBMP-2 onto plasma-sprayed hydroxyapatite implant surfaces by immersion in protein solution before implant installation would result in significantly improved bone apposition. Using a sheep iliac model, titanium (Ti) and plasma-sprayed calcium–phosphate (PSCaP)-coated implants uncoated and coated with rhBMP-2 were assessed for their osteogenic effects in the peri-implant area over time in terms of osseointegration and de novo bone formation. After 3 and 6 weeks postoperatively, the samples were retrieved and were subjected to bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) evaluation. When rhBMP-2 was applied to the PSCaP surface, significant increases in BIC and BAFO were observed at 3 weeks in vivo, whereas when adsorbed directly onto the titanium implant surface, rhBMP-2 did not as effectively improve the bone response (although significantly higher than control Ti). The outcomes of the present study suggested that the combination of plasma-sprayed calcium–phosphate surface and rhBMP-2 coating significantly enhanced osseointegration, which validated the postulated hypothesis.

Citation

(10.1002/jbm.a.34862)
Force-Clamp Experiments Reveal the Free-Energy Profile and Diffusion Coefficient of the Collapse of Protein Molecules

Abstract

We present force-clamp data on the collapse of ubiquitin polyproteins from a highly extended state to the folded length, in response to a quench in the force from 110 pN to 5 or 10 pN. Using a recent method for free-energy reconstruction from the observed nonequilibrium trajectories, we find that their statistics is captured by simple diffusion along the end-to-end length. The estimated diffusion coefficient of ∼100  nm2  s-1 is significantly slower than expected from viscous effects alone, possibly because of the internal degrees of freedom of the protein. The free-energy profiles give validity to a physical model in which the multiple protein domains collapse all at once and the role of the force is approximately captured by the Bell model.

Citation

(10.1103/PhysRevLett.110.128301)
Anisotropic nanocrystal arrays organized on protein lattices formed by recombinant clathrin fragments

Abstract

Recombinant clathrin protein fragments form assemblies that template gold nanocrystals in an array across the latticed surface. The nanocrystals exhibit unusual anisotropic morphologies with long range ordering, both of which are dependent upon the presence of a hexahistidine tag on the clathrin heavy chain fragments.

Citation

(10.1039/C2JM35019J)
Modulating Supramolecular Assemblies and Mechanical Properties of Engineered Protein Materials by Fluorinated Amino Acids

Abstract

Here we describe the biosynthesis and characterization of fluorinated protein block polymers comprised of the two self-assembling domains (SADs): elastin (E) and the coiled-coil region of cartilage oligomeric matrix proteins (C). Fluorination is achieved by residue-specific incorporation of p-fluorophenylalanine (pFF) to create pFF-EC, pFF-CE, and pFF-ECE. Global fluorination results in downstream effects on the temperature-dependent secondary structure, supramolecular assembly, and bulk mechanical properties. The impact of fluorination on material properties also differs depending on the orientation of the block configurations as well as the number of domains in the fusion. These studies suggest that integration of fluorinated amino acids within protein materials can be employed to tune the material properties, especially mechanical integrity.

Citation

(10.1021/bm3005116)
Effects of Divalent Metals on Nanoscopic Fiber Formation and Small Molecule Recognition of Helical Proteins

Abstract

Metal dependent protein-based assemblies derived from the cartilage oligomeric matrix protein (C) coiled-coil domain (His6-C) and two variants with mutation at position 40 (His6-T40A) and 44 (His6-L44A) are explored. All proteins have an N-terminal hexahistidine tag (His6) that interacts with divalent metal ions Zn(II) and Ni(II). Binding to Zn(II) confers enhanced helical structure and stability, while Ni(II) promotes aggregation. Surprisingly, His6-L44A undergoes a conformational switch from unstructured to α-helix in the presence of Zn(II). Both His6-C and His6-T40A further assemble into discrete nanofibers that appear to be stabilized by Zn(II) in which the fiber formation is dictated by the α-helical content. Because Ni(II) promotes aggregation, the proteins visibly cluster, forming large fiber mats in the case of His6-C and His6-T40A or aggregated structures as observed for His6-L44A. Due to the unique pentameric assembly of the proteins, recognition of a small molecule within the pore is assessed using curcumin as the guest molecule. In the presence of Zn(II), there is enhanced binding to curcumin, while the addition of Ni(II) causes a loss in binding. It is shown that metal binding serves as a trigger to control the conformation of the proteins, affecting the nanoscopic fibrous assemblies and small molecule recognition abilities.

Citation

(10.1002/adfm.201101627)
Characterization and identification of the protein partners of Fn3 domain in FnTm2

Abstract

Recently, a novel transmembrane protein was found to be up-regulated in the auditory learning pathway of birds and mammals. The protein, FnTm2, was predicted to have an extracellular fibronectin III (Fn3) domain and a single transmembrane domain. By contrast to other studied Fn3 domains the extracellular domain of FnTm2 bears several cysteine residues, which are predicted to form disulfide bonds. The Fn3 domain of the FnTm2 protein was expressed in DH5-α Escherichia coli (E. coli) cells, purified and characterized by circular dichroism (CD). In order to identify binding partners to Fn3, the isolated protein was incubated with bird brain lysate for a pull down treatment. Of the proteins recognized, myelin basic protein (MBP) was identified as a bona fide partner; it was further characterized for binding to Fn3 in vitro via fluorescence spectroscopy and confirmed via isothermal calorimetry (ITC).

Citation

(10.1016/j.pep.2011.08.026)
Artificial Protein Block Polymer Libraries Bearing Two SADs: Effects of Elastin Domain Repeats

Abstract

We have generated protein block polymer EnC and CEn libraries composed of two different self-assembling domains (SADs) derived from elastin (E) and the cartilage oligomeric matrix protein coiled-coil (C). As the E domain is shortened, the polymers exhibit an increase in inverse transition temperature (Tt); however, the range of temperature change differs dramatically between the EnC and CEn library. Whereas all polymers assemble into nanoparticles, the bulk mechanical properties of the EnC are very different from CEn. The EnC members demonstrate viscolelastic behavior under ambient conditions and assemble into elastic soft gels above their Tt values. By contrast, the CEn members are predominantly viscous at all temperatures. All library members demonstrate binding to curcumin. The differential thermoresponsive behaviors of the EnC and CEn libraries in addition to their small molecule recognition abilities make them suitable for potential use in tissue engineering and drug delivery.

Citation

(10.1021/bm201083d)
Modulating substrate specificity of histone acetyltransferase with unnatural amino acids

Abstract

Controlling the substrate specificity of enzymes is a major challenge for protein engineers. Here we explore the effects of residue-specific incorporation of ortho-, meta- and para-fluorophenylalanine (oFF, mFF, pFF) on the selectivity of human histone acetyltransferase (HAT) protein, p300/CBP associated factor (PCAF). Varying the position of the fluorine group in the phenylalanine ring confers different effects on the ability of PCAF to acetylate target histone H3 as well as non-histone p53. Surprisingly, pFF–PCAF exhibits an increase in activity for non-histone p53, while mFF–PCAF is selective for histone H3. These results suggest that global incorporation of unnatural amino acids may be used to re-engineer protein specificity.

Citation

(10.1039/C1MB05148B)
Identification and comparison of cutinases for synthetic polyester degradation

Abstract

Cutinases have been exploited for a broad range of reactions, from hydrolysis of soluble and insoluble esters to polymer synthesis. To further expand the biotechnological applications of cutinases for synthetic polyester degradation, we perform a comparative activity and stability analysis of five cutinases from Alternaria brassicicola (AbC), Aspergillus fumigatus (AfC), Aspergillus oryzae (AoC), Humicola insolens (HiC), and the well-characterized Fusarium solani (FsC). Of the cutinases, HiC demonstrated enhanced poly(ε-caprolactone) hydrolysis at high temperatures and under all pH values, followed by AoC and AfC. Both AbC and FsC are least stable and function poorly at high temperatures as well as at acidic pH conditions. Surface charge calculations and phylogenetic analysis reveal two important modes of cutinase stabilization: (1) an overall neutral surface charge within the “crowning area” by the active site and (2) additional disulfide bond formation. These studies provide insights useful for reengineering such enzymes with improved function and stability for a wide range of biotransformations.

Citation

(10.1007/s00253-011-3402-4)
Development and Implementation of High School Chemistry Modules Using Touch-Screen Technologies

Abstract

Technology was employed to motivate and captivate students while enriching their in-class education. An outreach program is described that involved college mentors introducing touch-screen technology into a high school chemistry classroom. Three modules were developed, with two of them specifically tailored to encourage comprehension of molecular bonding principles using a chemistry-based iPad app. Feedback-oriented lessons were utilized to pinpoint and address the students’ learning needs and preferences. Integration of the touch-screen technology with the chemistry curriculum demonstrated favorable results for all people involved: the high school teacher received assistance in the classroom, the college mentors gained experience as well as encouraged the high school students to further pursue chemical education, and the high school students received reinforcement in their chemistry curriculum.

Citation

(10.1021/ed200484n)
Enhanced Refoldability and Thermoactivity of Fluorinated Phosphotriesterase

Abstract

Teflon-like globular proteins: Global incorporation of p-fluorophenylalanine (pFF) in the S phosphotriesterase dimer (see figure) was found to stabilize the protein and led to refoldability and improved activity for substrates at elevated temperatures.

Citation

(10.1002/cbic.201100221)
Implementing and evaluating mentored Chemistry-Biology technology lab modules to promote early Interest in science

Abstract

Chemistry has become increasingly multidisciplinary. Starting in middle school and high school, however, the different disciplines are taught as distinct subjects and little effort is made to emphasize overlapping concepts. Development of innovative approaches to teaching science through the integration of technology is needed to provide students with a better learning experience that embraces multiple disciplines. Here, we describe an outreach and mentoring program between college and 7th grade students to: (i) encourage 7th grade students to pursue science and engineering degrees; (ii) provide the college students with mentoring experience; and (iii) assist teachers with state-of-the-art educational tools that enhance their teaching.

Citation

(10.1021/ed100476e)
College-Mentored Polymer/Materials Science Modules for Middle and High School Students

Abstract

Polymers are materials with vast environmental and economic ramifications, yet are generally not discussed in secondary education science curricula. We describe a program in which college mentors develop and implement hands-on, polymer-related experiments to supplement a standard, state regents-prescribed high school chemistry course, as well as a middle school elective course on polymers. Interactive experimentation and feedback-oriented design are highlighted as critical elements to the success of the program. The experiments have been executed in two vastly different institutions: a New York City magnet high school for underprivileged females, and a private middle school for privileged male and female students; the similarities and differences are juxtaposed.

Citation

(10.1021/ed1005618)
Mutagenesis of tGCN5 core region reveals two critical surface residues F90 and R140

Abstract

Tetrahymena General Control Non-Derepressor 5 (tGCN5) is a critical regulator of gene transcription via acetylation of histones. Since the acetylation ability has been attributed to the “core region”, we perform mutagenesis of residues within the tGCN5 “core region” in order to identify those critical for function and stability. Residues that do not participate in catalysis are identified, mutated and characterized for activity, structure and thermodynamic stability. Variants I107V, Q114L, A121T and A130S maintain the acetylation function relative to wild-type tGCN5, while variants F90Y, F112R and R140H completely abolish function. Of the three non-functional variants, since F112 is mutated into a non-homologous charged residue, a loss in function is expected. However, the remaining two variants are mutated into homologous residues, suggesting that F90 and R140 are critical for the activity of tGCN5. While mutation to homologous residue maintains acetylation of histone H3 for the majority of the variants, the two surface-exposed residues, F90 and R140, appear to be essential for tGCN5 function, structure or stability.

Citation

(10.1016/j.bbrc.2010.08.069)
Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis

Abstract

The HIV-1 Tat peptide has been successfully used for intracellular gene delivery. Likewise, various lipid-based methods have shown increased endocytosis and can influence endosomal escape. This study combines the favorable properties of Tat peptide with that of lipid systems for DNA delivery. We combined the lipid FuGENE HD (FH) with the Tat peptide sequence modified with histidine and cysteine residues (mTat). mTat/FH transfection was evaluated by luciferase expression plasmid in five cell types. mTat/FH produced significant improvement in transfection efficiency of all cell lines when compared to FH or mTat. Treatment with chloroquine, associated with energy-dependent endocytosis, significantly increased transfection efficiency with mTat/FH while incubation at low temperature decreased it. The zeta potential of mTat/FH/DNA was significantly higher compared to FH, mTat, or their DNA combination in the presence of serum, and it was correlated with transfection efficiency. The particle size of the FH/DNA complex was significantly reduced by addition of mTat. Filipin III, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/FH transfection, but transfection was increased by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. These findings demonstrated the feasibility of using a combination of mTat with lipids, utilizing temperature-dependent and caveolae-mediated endocytosis, as a potentially attractive non-viral gene vector.

Citation

(10.1016/j.jconrel.2011.02.004)
Mutagenesis of tGCN5 core region reveals two critical surface residues F90 and R140

Abstract

Tetrahymena General Control Non-Derepressor 5 (tGCN5) is a critical regulator of gene transcription via acetylation of histones. Since the acetylation ability has been attributed to the “core region”, we perform mutagenesis of residues within the tGCN5 “core region” in order to identify those critical for function and stability. Residues that do not participate in catalysis are identified, mutated and characterized for activity, structure and thermodynamic stability. Variants I107V, Q114L, A121T and A130S maintain the acetylation function relative to wild-type tGCN5, while variants F90Y, F112R and R140H completely abolish function. Of the three non-functional variants, since F112 is mutated into a non-homologous charged residue, a loss in function is expected. However, the remaining two variants are mutated into homologous residues, suggesting that F90 and R140 are critical for the activity of tGCN5. While mutation to homologous residue maintains acetylation of histone H3 for the majority of the variants, the two surface-exposed residues, F90 and R140, appear to be essential for tGCN5 function, structure or stability.

Citation

(10.1016/j.bbrc.2010.08.069)
Supramolecular assembly and small molecule recognition by genetically engineered protein block polymers composed of two SADs

Abstract

Genetically engineered protein block polymers are an important class of biomaterials that have gained significant attention in recent years due to their potential applications in biotechnology, electronics and medicine. The majority of the protein materials have been composed of at least a single self-assembling domain (SAD), enabling the formation of supramolecular structures. Recently, we developed block polymers consisting of two distinct SADs derived from an elastin-mimetic polypeptide (E) and the alpha-helical COMPcc (C). These protein polymers, synthesized as the block sequences—EC, CE, and ECE—were assessed for overall conformation and macroscopic thermoresponsive behavior. Here, we investigate the supramolecular assembly as well as the small molecule binding and release profile of these block polymers. Our results demonstrate that the protein polymers assemble into particles as well as fully or partially networked structures in a concentration dependent manner that is distinct from the individual E and C homopolymers and the E+C non-covalent mixture. In contrast to synthetic block polymers, the structured assembly, binding and release abilities are highly dependent on the composition and orientation of the blocks. These results reveal the promise for these block polymers for therapeutic delivery and biomedical scaffolds.

Citation

(10.1039/C002353A)
Biotransformations Using Cutinase

Abstract

There is a growing interest in sustainable and environmentally-friendly solutions for the industrial manufacturing of chemicals to replace non-renewable fossil fuel-based feedstocks. Biotransformations provide an alternative methodology to traditional reactions by taking advantage of the biochemical diversity of microorganisms to provide a chemo-, regio- and enantioselectivity, which are not always available via traditional synthetic approaches. Cutinases are enzymes secreted from phytopathogens and have been proven to be useful for several different biotransformations. This review will investigate the structure and function of cutinase. Further, it will detail cutinase activity towards both natural and non-natural substrates as well as methods employed to impart stability. Finally, we will describe the different role that cutinase has played in biotransformation reactions for biotechnology applications.

Citation

Baker, Peter James, and Jin Kim Montclare. “Biotransformations Using Cutinase.” In ACS symposium series, vol. 1043, pp. 141-158. Oxford University Press, 2010.
Incorporation of Unnatural Amino Acids for Synthetic Biology

Abstract

The challenge of synthetic biology lies in the construction of artificial cellular systems. This requires the development of modular “parts” that can be integrated into living systems to elicit an artificial, yet programmed, response or function. The development of methods to engineer proteins bearing unnatural amino acids (UAAs) provides essential components that may address this challenge. Here we review the emerging strategies for incorporating UAAs into proteins with the endgame of engineering artificial cells and organisms.

Citation

(10.1039/B909200P)
Structural and Functional Studies of Aspergillus oryzae Cutinase: Enhanced Thermostability and Hydrolytic Activity of Synthetic Ester and Polyester Degradation

Abstract

Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.

Citation

(10.1021/ja9046697)
Artificial Protein Block Copolymers Blocks Comprising Two Distinct Self-Assembling Domains

Abstract

It’s the way that you do it: Block polymers composed of elastin (E) and COMPcc (C) domains (see figure) have been synthesised and characterised. The protein motifs were chosen for their structures and distinct self-assembly modes. We demonstrate that the micro- and macrostructures of the polymers are dictated by the orientation of the fusions and the number of repeated blocks.

Citation

(10.1002/cbic.200900539)

N-terminal Aliphatic Residues Dictate the Structure, Stability, Assembly and Small Molecule Binding of the coiled-coil region of COMP

Abstract

The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D3 (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the α-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.

Citation

(10.1021/bi900534r)

Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity

Abstract

To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.

Citation

(10.1016/j.bmcl.2009.07.093)

Frozen Cyclohexane-in-Water Emulsion as a Sacrificial Template for the Synthesis of Multilayered Polyelectrolyte Microcapsules

Abstract

This paper reports the application of frozen cyclohexane-in-water emulsions as sacrificial templates for the fabrication of hollow microcapsules through layer-by-layer assembly of polyelectrolytes, poly(styrenesulfonate sodium salt), and poly(allylamine hydrochloride). Extraction of the cyclohexane phase from frozen emulsions stabilized with 11 polyelectrolyte layers by compatibilization with 30% v/v ethanol leads to the formation of water-filled microcapsules while preserving the spherical geometry. The majority of microcapsules (>90%) are prepared with intact polyelectrolyte membranes as measured by their deformation induced by osmotic pressure. This work provides a new route for the synthesis of hollow multilayered microcapsules under mild operating conditions.

Citation

(10.1021/la901020j)

Biosynthesis and Stability of Coiled-Coil Peptides Containing (2S,4R)-5,5,5-Trifluoroleucine and (2S,4S)-5,5,5-Trifluoroleucine

Abstract

Life in stereo: We report the effects of 5,5,5-trifluoroleucine (TFL) stereochemistry on coiled-coil peptide biosynthesis and stability. We demonstrate that two diastereoisomers of TFL are activated and incorporated into peptides expressed in E. coli. Coiled-coil homodimers of these peptides exhibited increased stability. An equimolar mixture of the two fluorinated peptides formed a heterodimer of modestly enhanced thermal stability relative to the homodimers.

Citation

(10.1002/cbic.200800164)

Elastin-Based Protein Polymers

Abstract

The synthesis of well-defined macromolecular structures with controlled properties is critical for the production of advanced materials for biological and industrial applications. Inspired by nature’s ability to create proteins with exquisite control, we focus on the applications of elastin and elastin-derived polymers for materials design. The elucidation of elastin biochemical, conformational and physical properties offers insight into the fabrication of novel biomaterials. As part of this review, we highlight some of the recent advances that permit the generation of customized elastin-based polymers. These developments provide an added level of control vital to the future construction of tailor-made supramolecular structures with emergent physical, mechanical and biological properties.

Citation

(10.1021/bk-2008-0999.ch003)

Assembly of bioinspired helical protein fibers

Abstract

Advances in protein and peptide technologies not only enable the study of basic folding and function of natural structures but also the design of novel scaffolds with the ability to form assemblies of varied shapes and sizes. Tremendous progress has been made in our understanding of α-helices in nature especially in the context of the coiled-coil. The information gleaned from investigating coiled- coils has been used to design novel α-helical fibers with prescribed morphology and dimensions. This review focuses on the lessons learned from the assembly of natural coiled-coils and how this knowledge can be used to tailor helical fibers as novel bioinspired materials.

Citation

(10.1002/pat.1136)

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: Improved thermostability by a single-isoleucine mutant

Abstract

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

Citation

(10.1016/j.bmcl.2007.07.107)

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Abstract

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5′,5′,5′-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.

Citation

(10.1002/bit.20910)

Evolving Proteins of Novel Composition

Abstract

Changing its nature: Global incorporation of noncanonical amino acids can alter the behavior of proteins in useful ways. In some cases, however, replacement of natural amino acids by noncanonical analogues (blue bars in picture) can cause loss of protein stability. After several generations, functional proteins of non-natural composition were prepared through residue-specific incorporation combined with directed evolution.

Citation

(10.1002/ange.200600088)