Protein biomaterials have been used for a wide range biomedical applications due to their intrinsic biocompatibility, versatility and higher order structure. Multifunctional theranostic agents fabricated by incorporating the diagnostic modalities and drug payloads in protein nanoformulations have garnered significant attention in recent years. Protein-based theranostic agents manifest high target specificity, enhanced blood circulation and reduce reticuloendothelial system elimination. This review focuses on the fabrication of peptide- and protein-based nanoformulations for imaging guided therapy.
Punia, K.; Kronenberg, J. B.; Montclare, J. K. Mol. Sys. Des. Eng. 2019, 4, 1074–1094. doi: 10.1039/C9ME00143C
Keeping high school students engaged and motivated to learn complex scientific concepts can be difficult and challenging; this is especially true if the task feels daunting and unfamiliar to the students. Incorporating educational technology, such as KAHOOT, into the classroom can help students learn scientific material even when it is difficult. Our objective is to determine the effectiveness of gamification in an Advanced Placement Biology (AP biology) classroom by using the online game ‘KAHOOT!’ as a supplement to traditional teacher-centered learning. In addition, we determine the use of ‘KAHOOT!’ in enhancing student engagement and the learning experience for biology. A presentation on Transcription and Translation was given to a small group of high school AP Biology students (n = 18, 18 women). After the presentation, the students were given 15 questions and twenty seconds to answer each question in the ‘KAHOOT!’ game. Both the students and the teacher were given a post-activity survey to assess their interest in the activity. Based on the responses in the Students’ Survey, ‘KAHOOT!’ can be used as a gamified assessment tool to help students learn the topic of ‘Transcription and Translation’ by actively engaging them in a fun and exciting manner. The overall activity had a positive impact on the students and teacher as the students enjoyed learning Transcription and Translation through the use of ‘KAHOOT!’.
Jones, S. M.; Katyal, P.; Xie, X.; Nicolas, M. P.; Leung, E. M.; Noland, D. M.; Montclare, J. K. A ‘KAHOOT!’ Approach: The Effectiveness of Game-Based Learning for an Advanced Placement Biology Class.
Simul. Gaming 2019,
50 (6), 832–847.
doi：10.1177/1046878119882048
Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here, we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein-phase behavior and that of synthetic polymer systems.
Hill, L. K.; Meleties, M.; Katyal, P. ; Xie, X.; Delgado-Fukushima, E.; Jihad, T.; Liu, C. -F.; O’Neill, S.; Tu, R. S.; Renfrew, P. D.; Bonneau, R.; Wadghiri, Y. Z.; Montclare, J. K. Thermoresponsive Protein-Engineered Coiled-Coil Hydrogel for Sustained Small Molecule Release.
Biomacromolecules 2019,
20 (9), 3340–3351.
doi: 10.1021/acs.biomac.9b00107
Considerable effort has been devoted to generating novel protein- and peptide-based nanomaterials with their applications in a wide range of fields. Specifically, the unique property of proteins to self-assemble has been utilized to create a variety of nanoassemblies, which offer significant possibilities for next-generation biomaterials. In this minireview, we describe self-assembled protein- and peptide-based nanomaterials with focus on nanofibers and nanoparticles. Their applications in delivering therapeutic drugs and genes are discussed.
Katyal, P.; Meleties, M.; Montclare, J. K. Self-Assembled Protein- and Peptide-Based Nanomaterials.
ACS Biomater. Sci. Eng. 2019,
5 (9), 4132–4147.
doi: 10.1021/acsbiomaterials.9b00408
Proteins are versatile macromolecules that can perform a variety of functions. In the past three decades, they have been commonly used as building blocks to generate a range of biomaterials. Owing to their flexibility, proteins can either be used alone or in combination with other functional molecules. Advances in synthetic and chemical biology have enabled new protein fusions as well as the integration of new functional groups leading to biomaterials with emergent properties. This review discusses protein‐engineered materials from the perspectives of domain‐based designs as well as physical and chemical approaches for crosslinked materials, with special emphasis on the creation of hydrogels. Engineered proteins that organize or template metal ions, bear noncanonical amino acids (NCAAs), and their potential applications, are also reviewed.
Wang, Y.; Katyal, P.; Montclare, J. K. Protein-Engineered Functional Materials.
Adv. Healthc. Mater. 2019,
8, 1801374.
doi: 10.1002/adhm.201801374
Engineered proteins provide an interesting template for designing fluorine-19 (¹⁹F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed “fluorinated thermoresponsive assembled protein” (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting ¹⁹F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin–spin relaxation (T2) times and unaffected spin–lattice relaxation (T1) times. The nearly unchanging T1 relaxation rates and linearly dependent T2 relaxation rates have allowed for detection via zero echo time ¹⁹F MRI, and the in vivo MR potential has been preliminarily explored using ¹⁹F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive ¹⁹F MRI/MRS-traceable theranostic agents.
Hill, L. K.; Frezzo, J. A.; Katyal, P.; Hoang, D. H.; Gironda, Z. B. Y.; Xu, C.; Xie, X.; Delgado-Fukushima, E.; Wadghiri, Y. Z.; Montclare, J. K. Protein-Engineered Nanoscale Micelles for Dynamic ¹⁹F Magnetic Resonance and Therapeutic Drug Delivery.
ACS Nano. 2019,
13 (3), 2969–2985.
doi: 10.1021/acsnano.8b07481
We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells.
Kasai, H.; Inoue, K. Imamura, K. Yuvienco, C.; Montclare, J. K.; Yamano, S. J.Nanobiotechnology 2019, 17 (1), 11. doi: 10.1186/s12951-019-0444-8
Recombinant methods have been used to engineer artificial protein triblock polymers composed of two different self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering.
Yin, L.; Agustinus, A. S.; Yuvienco, C.; Minashima, T.; Schnabel, N. L.; Kirsch, T.; Montclare, J. K. Engineered Coiled-Coil Protein for Delivery of Inverse Agonist for Osteoarthritis. Biomacromolecules 2018, 19 (5), 1614–1624. doi: 10.1021/acs.biomac.8b00158
Recombinant methods have been used to engineer artificial protein triblock polymers composed of two different self-assembling domains (SADs) bearing one elastin (E) flanked by two cartilage oligomeric matrix protein coiled-coil (C) domains to generate CEC. To understand how the two C domains improve small molecule recognition and the mechanical integrity of CEC, we have constructed CL44AECL44A, which bears an impaired CL44A domain that is unstructured as a negative control. The CEC triblock polymer demonstrates increased small molecule binding and ideal elastic behavior for hydrogel formation. The negative control CL44AECL44A does not exhibit binding to small molecule and is inelastic at lower temperatures, affirming the favorable role of C domain and its helical conformation. While both CEC and CL44AECL44A assemble into micelles, CEC is more densely packed with C domains on the surface enabling the development of networks leading to hydrogel formation. Such protein engineered triblock copolymers capable of forming robust hydrogels hold tremendous promise for biomedical applications in drug delivery and tissue engineering.
Olsen, A. J.; Katyal, P.; Haghpanah, J. S.; Kubilius, M. B.; Li, R.; Schnabel, N. L.; O’Neill, S. C.; Wang, Y.; Dai, M.; Singh, N.; Tu, R. S.; Montclare, J. K. Protein Engineered Triblock Polymers Composed of Two SADs: Enhanced Mechanical Properties and Binding Abilities. Biomacromolecules 2018, 19 (5), 1552–1561. doi: https://doi.org/10.1021/acs.biomac.7b01259
My family emigrated from Korea, and I was born and raised in the Bronx. I was taught to value education, and my parents worked several jobs just so my brother and I could go to good schools. I loved math and science all throughout my life and got to experience research during my high school career. My love was fostered by my teachers, and, while I never considered an academic scientist as a career, it was my teachers and mentors who led me on my path.
Montclare, J. K. Family Matters: How Family Influenced My Career. In Mom the Chemistry Professor; Woznack, K. Charlebois, A., Cole R., Marzabadi C., Webster G., Eds.; Springer: Cham, Germany, 2018; pp 345–353. doi: 10.1007/978-3-319-78972-9_25
Tremendous effort has been dedicated to the design and assembly of bioinspired protein-based architectures with potential applications in drug delivery, tissue engineering, biosensing, and bioimaging. Here, we describe our strategy to generate fibers and bionanocomposites using the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc). Our construct, Q, engineered by swapping particular regions of COMPcc to optimize surface charge, self-assembles to form nanofibers. The Q protein nanofibers can efficiently bind curcumin to form robust mesofibers that can be potentially used for drug delivery and biomedical applications. In addition, using the same Q protein, we describe the biotemplation of gold nanoparticles (AuNP) in the presence and absence of the hexahistidine tag (His-tag). The Q bearing His-tag·AuNP (Q·AuNP) readily deposits on electrode surfaces, while Q without His-tag·AuNP (Qx·AuNP) stabilizes the soluble protein·gold bionanocomposites for several days without aggregating.
Katyal, P. and Montclare, J. K. Design and characterization of fibers and bionanocomposites using the coiled-coil domain of cartilage oligomeric matrix protein. In Protein Scaffolds. Methods in Molecular Biology, vol 1798; Humana Press: New York, NY, USA, 2018; pp. 239–263. doi: 10.1007/978-1-4939-7893-9_19
The effectiveness of blended learning was evaluated through the integration of an online chemistry platform, LabLessons. Two modules, Formation of Hydrogen and Titration, were designed by college mentors alongside classroom chemistry teachers to engage and allow high school students to better comprehend these scientific topics. The pre-lab modules introduced the students to experiments they were expected to perform in class the following day. The modules consisted of an introduction as well as either a visualization and/or simulation specific to each topic. Students and teachers who utilized LabLessons were surveyed to establish a preliminary research on the use of technology in classrooms. Student and teacher surveys demonstrated LabLessons to be an interactive and helpful tool to improve students’ understanding of conceptual ideas.
Jihad, T.; Klementowicz, E.; Gryczka, P.; Sharrock, C.; Maxfield, M.; Lee, Y.; Montclare, J. K. Perspectives on blended learning through the on-line platform, lablessons, for chemistry.
J. Technol. Sci. Educ. 2018,
8 (1), 34–44.
doi: 10.3926/jotse.312
An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000. When the lipoproteoplex is loaded with doxorubicin, the improved doxorubicin loading (∼40 μg Dox/mg CSP) results in a substantial decrease in MCF-7 cell viability.
Liu, C. F.; Chen, R.; Frezzo, J. A.; Katyal, P.; Hill, L. K.; Yin, L.; Srivastava, N.; More, H. T.; Renfrew, P. D.; Bonneau, R.; Montclare, J. K. Efficient Dual siRNA and Drug Delivery Using Engineered Lipoproteoplexes. Biomacromolecules 2017, 18 (9), 2688–2698. doi: 10.1021/acs.biomac.7b00203
As unique biopolymers, proteins can be employed for therapeutic delivery. They bear important features such as bioavailability, biocompatibility, and biodegradability with low toxicity serving as a platform for delivery of various small molecule therapeutics, gene therapies, protein biologics and cells. Depending on size and characteristic of the therapeutic, a variety of natural and engineered proteins or peptides have been developed. This, coupled to recent advances in synthetic and chemical biology, has led to the creation of tailor-made protein materials for delivery. This review highlights strategies employing proteins to facilitate the delivery of therapeutic matter, addressing the challenges for small molecule, gene, protein and cell transport.
Yin, L.; Yuvienco, C.; Montclare, J. K. Protein based therapeutic delivery agents: Contemporary developments and challenges. Biomaterials 2017, 134, 91–116. doi: 10.1016/j.biomaterials.2017.04.036
Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1, key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing siKeap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.
Rabbani, P. S.; Zhou, A.; Borab, Z. M.; Frezzo, J. A.; Srivastava, N.; More, H. T.; Rifkin, W. J.; David, J. A.; Berens, S. J.; Chen, R.; Hameedi, S.; Junejo, M. H.; Kim, C.; Sartor, R. A.; Liu, C. F.; Saadeh, P. B.; Montclare, J. K.; Ceradini, D. J. Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing. Biomaterials 2017, 132, 1–15. doi: 10.1016/j.biomaterials.2017.04.001
We explore the significance of phenylalanine outside of the phosphotriesterase (PTE) dimer interface through mutagenesis studies and computational modeling. Previous studies have demonstrated that the residue-specific incorporation of para-fluorophenylalanine (pFF) into PTE improves stability, suggesting the importance of phenylalanines in stabilization of the dimer. However, this comes at a cost of decreased solubility due to pFF incorporation into other parts of the protein. Motivated by this, eight single solvent-exposed phenylalanine mutants are evaluated via ROSETTA and good correspondence between experiments and these predictions is observed. Three residues, F304, F327, and F335, appear to be important for PTE activity and stability, even though they do not reside in the dimer interface region or active site. While the remaining mutants do not significantly affect structure or activity, one variant, F306L, reveals improved activity at ambient and elevated temperatures. These studies provide further insight into role of these residues on PTE function and stability.
Olsen, A. J.; Halvorsen, L. A.; Yang, C. -Y.; Ventura, R. B.; Yin, L.; Renfrew, P. D.; Bonneau, R.; Montclare, J. K. Impact of phenylalanines outside the dimer interface on phosphotriesterase stability and function. Mol. BioSyst. 2017, 13, 2092–2106. doi: 10.1039/c7mb00196g
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.
Frezzo, J. A. and Montclare, J. K. Natural Composite Systems for Bioinspired Materials. In Protein-based Engineered Nanostructures. Advances in Experimental Medicine and Biology, vol 940; Cortajarena, A., Grove, T. Eds.; Springer: Cham, Germany, 2016; pp 143–166. doi: 10.1007/978-3-319-39196-0_7
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.
Gryczka, P.; Klementowicz, E.; Sharrock, C.; Maxfield, M.; Montclare, J. K. LabLessons: Effects of Electronic Prelabs on Student Engagement and Performance. J. Chem. Educ. 2016, 93 (12), 2012–2017. doi: 10.1021/acs.jchemed.6b00394
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.
Gryczka, P.; Klementowicz, E.; Sharrock, C.; Montclare, J. Interactive online physics labs increase high school students’ interest.
J. Technol. Sci. Educ. 2016,
6 (3), 166–187.
doi: 10.3926/jotse.191
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
Dai, M.; Frezzo, J. A. Sharma, E.; Chen, R.; Singh, N.; Yuvienco, C.; Caglar, E.; Xiao, S.; Saxena, A.; Montclare, J. K. Engineered Protein Polymer-Gold Nanoparticle Hybrid Materials for Small Molecule Delivery.
J. Nanomed. Nanotechnol. 2016,
7 (1), 356.
doi: 10.4172/2157-7439.1000356
Over the course of evolution, nature has made extensive use of the α-helical coiled-coil for a wide breadth of applications in gene regulation, mitosis, cellular and muscular motility as well as cytoskeletal structure. An estimated 2-10% of all proteins harbor the coiled-coil motif, demonstrating the versatility of this particular motif across the spectrum of protein functions. While the functional diversity of the coiled-coil motif in nature is well established, there are expanding roles these motifs may play in tissue engineering and drug delivery. Before delving into this promising field of coiled-coil proteins as drug delivery vehicles, it is pertinent to discuss the underlying principles that govern the assembly of the coiled-coil.
Frezzo, J. A. and Montclare, J. K. Exploring the potential of engineered coiled-coil protein microfibers in drug delivery.
Ther. Deliv. 2015,
6 (6), 643–646.
doi: 10.4155/tde.15.19
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.
Hume, J.; Chen, R.; Jacquet, R.; Yang, M.; Montclare, J. K. Tunable Conformation-Dependent Engineered Protein·Gold Nanoparticle Nanocomposites.
Biomacromolecules 2015,
16 (6), 1706–1713.
doi: 10.1021/acs.biomac.5b00098
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.
More, H. T.; Zhang, K. S.; Srivastava, N.; Frezzo, J. A.; Montclare, J. K. Influence of Fluorination on Protein-Engineered Coiled-Coil Fibers.
Biomacromolecules 2015,
16 (4), 1210–1217.
doi: 10.1021/bm5019062
Students have access to the Internet at their fingertips via e-tablets and smart phones. However, the STEM fields are struggling to remain relevant in students’ lives outside the classroom. In an effort 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 utilized touch screen technology in conjunction with hands-on experiments and traditional lecturing to create an entertaining, relevant, and effective classroom experience.
Chacko, P.; Appelbaum, S.; Kim, H.; Zhao, J.; Montclare, J. K. Integrating Technology in STEM Education.
J. Technol. Sci. Educ. 2015,
5 (1), 5–14.
doi: 10.3926/jotse.124
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.
Kim, H.; Chacko, P.; Zhao, J.; Montclare, J. K. Using Touch-Screen Technology, Apps, and Blogs To Engage and Sustain High School Students’ Interest in Chemistry Topics.
J. Chem. Educ. 2014,
91 (11), 1818–1822.
doi: 10.1021/ed500234z
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 https://www.fiercepharma.com/drug-delivery/nyu-researchers-break-nano-barrier-to-engineer-first-protein-microfiber
Hume, J.; Sun, J.; Jacquet, R.; Renfrew, P. D.; Martin, J. A.; Bonneau, R.; Gilchrist, M. L.; Montclare, J. K. Engineered Coiled-Coil Protein Microfibers.
Biomacromolecules 2014,
15 (10), 3503–3510.
doi: 10.1021/bm5004948
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 https://www.medicalnewstoday.com/articles/280450
Yang, C. -Y.; Renfrew, P. D.; Olsen, A. J.; Zhang, M.; Yuvienco, C.; Bonneau, R.; Montclare, J. K. Improved Stability and Half-Life of Fluorinated Phosphotriesterase Using Rosetta.
ChemBioChem 2014,
15 (12), 1761–1764.
doi: 10.1002/cbic.201402062
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.
More, H. T.; Frezzo, J. A.; Dai, J.; Yamano, S.; Montclare, J. K. Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex.
Biomaterials 2014,
35 (25), 7188–7193.
doi: 10.1016/j.biomaterials.2014.05.005
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.
Yamano, S.; Dai, J.; Hanatani, S.; Haku, K.; Yamanaka, T.; Ishioka, M.; Takayama, T.; Yuvienco, C.; Khapli, S.; Moursi, A. M.; Montclare, J. K.
Biomaterials 2014,
35 (5),1705–1715.
doi: 10.1016/j.biomaterials.2013.11.012
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.
[cite]10.1021/bm401304w[/cite]
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.
Yoo, D.; Tover, N.; Jimbo, R.; Marin, C.; Anchieta, R. B.; Machado, L. S.; Montclare, J.; Guastaldi, F. P. S.; Janal, M. N.; Coelho, P. G. Increased osseointegration effect of bone morphogenetic protein 2 on dental implants: An in vivo study.
J. Biomed. Mater. Res. 2014, 102 (6), 1921–1927.
doi: 10.1002/jbm.a.34862
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.
Lannon, H.; Haghpanah, J. S.; Montclare, J. K.; Vanden-Eijnden, E.; Brujic, J. Force-Clamp Experiments Reveal the Free-Energy Profile and Diffusion Coefficient of the Collapse of Protein Molecules.
Phys. Rev. Lett. 2013,
110, 128301.
doi: 10.1103/PhysRevLett.110.128301
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.
Hom, N.; Mehta, K. R.; Chou, T.; Foraker, A. B.; Brodsky, F. M.; Kirshenbaum, K.; Montclare, J. K. Anisotropic nanocrystal arrays organized on protein lattices formed by recombinant clathrin fragments.
J. Mater. Chem.,
2012,
22, 23335–23339.
doi: 10.1039/C2JM35019J
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.
Yuvienco, C.; More, H. T.; Haghpanah, J. S.; Tu, R. S.; Montclare, J. K. Modulating Supramolecular Assemblies and Mechanical Properties of Engineered Protein Materials by Fluorinated Amino Acids.
Biomacromolecules 2012,
13 (8), 2273–2278.
doi: 10.1021/bm3005116
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.
[cite]10.1002/adfm.201101627[/cite]
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).
Gunasekar, S. K.; Anjia, L.; Matsui, H.; Montclare, J. K. Effects of Divalent Metals on Nanoscopic Fiber Formation and Small Molecule Recognition of Helical Proteins.
Adv. Funct. Mater. 2012,
22 (10), 2154–2159.
doi: 10.1016/j.pep.2011.08.026
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.
Baker, P. J.; Poultney, C.; Liu, Z.; Gross, R.; Montclare, J. K. Identification and comparison of cutinases for synthetic polyester degradation.
Appl. Microbiol. Biotechnol. 2012,
93, 229–240.
doi: 10.1007/s00253-011-3402-4
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.
Dai, M.; Haghpanah, J.; Singh, N.; Roth, E. W.; Liang, A.; Tu, R. S.; Montclare, J. K. Artificial Protein Block Polymer Libraries Bearing Two SADs: Effects of Elastin Domain Repeats.
Biomacromolecules 2011,
12 (12), 4240–4246.
doi: 10.1021/bm201083d
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.
Mehta, K. R.; Yang, C. Y.; Montclare, J. K. Modulating substrate specificity of histone acetyltransferase with unnatural amino acids.
Mol. BioSyst.,
2011,
7, 3050–3055.
doi: 10.1039/C1MB05148B
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.
Lewis, M. S.; Zhao, J.; Montclare, J. K. Development and Implementation of High School Chemistry Modules Using Touch-Screen Technologies.
J. Chem. Educ. 2012,
89 (8), 1012–1018.
doi: 10.1021/ed200484n
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.
Baker, P. J. and Montclare, J. K. Enhanced Refoldability and Thermoactivity of Fluorinated Phosphotriesterase.
ChemBioChem 2011,
12 (12), 1845–1848.
doi: 10.1002/cbic.201100221
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.
Chan, Y. M.; Hom, W.; Montclare, J. K. Implementing and Evaluating Mentored Chemistry−Biology Technology Lab Modules To Promote Early Interest in Science.
J. Chem. Educ. 2011,
88 (6), 751–754.
doi: 10.1021/ed100476e
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.
Lorenzini, R. G.; Lewis, M. S.; Montclare, J. K. College-Mentored Polymer/Materials Science Modules for Middle and High School Students.
J. Chem. Educ. 2011,
88 (8), 1105–1108.
doi: 10.1021/ed1005618
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.
Yamano, S.; Dai, J.; Yuvienco, C.; Khapli, S.; Moursi, A. M.; Montclare, J. K. Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis.
J. Control. Release 2011,
152 (2), 278–285.
doi: 10.1016/j.jconrel.2011.02.004
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.
Mehta, K. R.; Chan, Y. M.; Lee, M. X.; Yang, C. Y.; Voloshchuka, N.; Montclare, J. K. Mutagenesis of tGCN5 core region reveals two critical surface residues F90 and R140.
Biochem. Biophys. Res. Commun. 2010,
400 (3), 363–368.
doi: 10.1016/j.bbrc.2010.08.069
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.
Haghpanah, J. S.; Yuvienco, C.; Roth, E. W.; Liang, A.; Tu, R. S.; Montclare, J. K. Supramolecular assembly and small molecule recognition by genetically engineered protein block polymers composed of two SADs.
Mol. BioSyst. 2010,
6, 1662–1667.
doi: 10.1039/C002353A
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.
Baker, P. J. and Montclare, J. K. Biotransformations Using Cutinase. In
Green Polymer Chemistry: Biocatalysis and Biomaterials ACS symposium series, vol. 1043; American Chemical Society: Washington, DC, 2010; pp. 141-158.
doi: 10.1021/bk-2010-1043.ch011
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.
Voloshchuka, N. and Montclare, J. K. Incorporation of unnatural amino acids for synthetic biology.
Mol. BioSyst. 2010,
6, 65–80.
doi: 10.1039/B909200P
