Biomineralization, Bio-Materials and Nano-Materials Chemistry
Not ACCEPTING NEW GRAD STUDENTS
B.A. University of California, San Diego, 1986;
Ph.D., Cornell University, 1991;
Postdoctoral, University of Bath (UK) 1992-1994
· BCH 580 SPECIAL TOPICS IN BIOCHEMISTRY
· CHMY 401 ADVANCED INORGANIC CHEMISTRY
Awards and Professional Activities:
College of Letters and Sciences Distinguished Professor, Montana State University (2008)
Charles and Nora L. Wiley Award for Meritorious Research, Montana State University (2005)
University Merit Award for Research (1999)
Outstanding Faculty Award for Teaching (1996)
Joseph E. Meyer Award for Undergraduate Research, UCSD (1986)
Natural Supramolecular Architectures:
Virus Capsids for Biomimetic and Bio-inspired Materials Chemistry
Fig 1. The supramolecular architectures of icosahedral virus capsids are amenable to modifications [Ref 1]
Fig 2. The four morphological forms of the P22 capsid.
My research group has pioneered the use of viruses as supramolecular platforms for synthetic manipulation with a range of applications from materials to medicine [1-3]. Through understanding the inherent properties of these cage-like architectures, which include high symmetry and self-assembly, we have exploited their use as synthetic templates for modification and molecular design. An appreciation of these properties has resulted in a paradigm shift from the study of viruses as purely disease causing agents to highly useful supramolecular assemblies, which can be chemically and genetically modified. Future synthetic manipulation can impart new function to these architectures, combining the best of evolution and directed synthetic design. This work is providing new avenues of exploration in a variety of areas and is exciting for its interdisciplinary impact and applications.
Supramolecular virus architectures can be viewed as molecular containers with three distinct interfaces that impart function (and can be independently manipulated synthetically). These are: the exterior surface, the interior surface, and the interface between the subunits that make up the overall architecture (Fig 1 - modification can take place on either the interior or exterior interface or at the interface between the subunit building blocks). These protein cage architectures have been used as constrained reaction environments for the synthesis and sequestration of nanomaterials and the encapsulation of therapeutic drugs, and diagnostic imaging agents, and active proteins encapsulated within the cage. The interior surfaces can be manipulated to direct encapsulation of a cargo, while the outer surfaces have been used to incorporate targeting moieties to direct and target specific surfaces including cells/tissues and solid supports. The interface between subunits can be influenced to promote disassembly, and reassembly into a wide range of alternate architectural assemblies. Current work in the lab is described below and is focused on developing both chemical and genetic methods for directed synthesis to alter the physical properties of the modified capsid architecture.
Most of the current, funded, projects in the lab are centered on our use of the capsid derived from the bacteriophage P22 [4-6], which we have found to be structurally robust, readily amenable to chemical and genetic manipulation, and easy (and safe) to produce in large quantities. This supramolecular architecture self-assembles into a homogeneous â€˜procapsidâ€™ structure that has been structurally characterized by high-resolution cryo-TEM with near atomic level structural models, which allow us to rationally (re)design the capsid. The P22 capsid is capable of undergoing a series of controlled heat induced structural transformations (Fig 2 Procapsid (PC) - assembled from coat protein (CP) with scaffold protein (SP) encapsulated inside. Empty Shell (ES) with the SP removed from the PC. Expanded Shell (EX) after heat treated to 65ÂºC. Wiffle Ball (WB), heating to 75ÂºC results in the loss of pentamers and the formation of large pores through the capsid shell.) to create a series of extremely stable structures, which differ in size, cargo-loading, and porosity of the capsid shell. The P22 capsid is a highly versatile nano-container with a large internal cavity ideal for high-capacity loading of polymers, drugs, inorganic nanoparticles, polypeptides and active proteins.
1. T. Douglas and M. Young:
Science, 2006, 312, 873-875.
2. S. Kang and T. Douglas:
Science, 2010, 327, 42-43
3. T. Douglas and M. Young:
Nature, 1998, 393, 152-155.
S. Kang, M. Uchida, A. O'Neil, R. Li, P. E. Prevelige and T. Douglas:
Biomacromolecules, 2010, 11, 2804-2809.
A. O'Neil, C. R. Reichhardt, P. E. Prevelige and T. Douglas:
Angewandte Chemie, 2011, 50, 7425-7428.
C. R. Reichhardt, A. O'Neil, M. Uchida, P. E. Prevelige and T. Douglas:
Chemical Communications, 2011, 47, 6326-6328.
Synthesis, Structure, Spectroscopy, Protein Chemistry, Physical, Mechanism, Inorganic, Chemical Biology, Biophysical, Bioinorganic, Biochemistry
Constrained Polymer synthesis, using the P22 capsid as a template
Fig 3. Formation of functionalized ATRP monomers and examples of metal chelating monomers synthesized
The use of protein-polymer composites for materials and medical applications aims to take advantage of the exquisite monodispersity and bioactivity of biomacromolecules while imparting new materials properties via polymer conjugation. By conjugating polymers to the biomolecule, the composite material can exhibit improved retention and lowered immunogenicity, in addition to materials properties such as light, pH, and thermal responsiveness.
Viral capsids provide an ideal template for the synthesis of these hybrid polymer materials. Internal polymerization dramatically increases the surface area and through polymer chain interaction (covalent or non-covalent) changes in the physical properties of the protein cage (thermal stability, stiffness) can be affected. Polymerization on the exterior of the protein cage provides a â€˜polymer-brushâ€™-like architecture where the interaction between particles can be designed to facilitate non-covalent interactions as a means to initiate hierarchical assembly or to screen interactions of these protein cage-polymer composites.
We are exploring four complementary types of polymer syntheses (Fig 3). These are: (1) atom transfer radical polymerization (ATRP), (2) azide-alkyne [3+2] â€˜clickâ€™ polymerization; (3) coordination polymerization; (4) genetically programmed polypeptide synthesis. ATRP provides a mild, very rapid, and selective means of generating polymer/copolymer chains covalently attached to the protein interface. â€œClick chemistryâ€ when coupled to the protein is an effective and gentle means to grow a variety of functional polymers in a step-wise manner . Coordination polymers can be constructed inside the protein cage via metal-chelate interactions (self assembly of MOF-like structures). Also, incorporation of metal chelating monomers into either the ATRP or â€˜clickâ€™ synthesis allows us to explore a combination of these polymerization approaches. Incorporation and encapsulation of designed polypeptides on the interior of the P22 (described below - Project 3) is an exciting parallel to the chemical polymer syntheses.
We have shown that the formation of crosslinked polymer networks, inside our protein cages, using azide-alkyne click chemistry, results in a confined hyperbranched polymer scaffold . This polymer network alters the physical properties of the protein cage and, with appropriate design of the monomers, the polymer side-chains act as attachment points for functional molecules of interest. When Gd-chelates are appended to the polymer, the construct can be used as a contrast agent with vastly improved relaxivity per particle (see MRI contrast agent section) . Alternatively, if the monomer is based on a coordination complex (with polymerizable sites on the ligand), the resulting coordination polymer (metallopolymer) incorporates the metal centers as part of the polymer . This approach to coordination polymer formation allows for the incorporation of metal complexes into the protein-polymer composite that are stable in aqueous conditions, but may not be readily formed under protein compatible conditions.
Using ATRP allows us to form the desired polymer in a continuous process and has the advantage of fast syntheses. This method results in products with low polydispersity, but is also promiscuous with respect to the range of monomers and functional groups that can be included in the polymer. When modified metal chelators (imidazole, pyridine, iminodiacetic acid, catecholâ€¦) are incorporated into the acrylate or acrylamide monomers the resulting polymer can be doped with appropriate metals forming extensively cross-linked structures similar to those found in biological tissues. If the monomer bears a primary amine, or other addressable group, then coordination complexes or other molecules of interest (drugs, fluorophores, light harvesting chromophores, reactive metal coordination complexesâ€¦) can be appended to the polymer introducing new desired functionalities. In addition to development of highly promising MRI contrast agents we are currently exploring functionalized polymer packaging inside P22 as a means to affect reactive site proximity (coupled light harvesting and H2 production) and probing the effects of molecular crowding on reactivity.
1. M. J. Abedin, L. Liepold, P. Suci, M. Young and T. Douglas:
Journal of the American Chemical Society, 2009, 131, 4346-4354.
2. L. O. Liepold, M. J. Abedin, E. D. Buckhouse, J. A. Frank, M. J. Young and T. Douglas:
Nano Letters, 2009, 9, 4520-4526.
3. J. Lucon, M. J. Abedin, M. Uchida, L. Liepold, C. C. Jolley, M. Young and T. Douglas:
Chemical Communications, 2010, 46, 264-266.
Synthesis, Protein Chemistry, Inorganic, Chemical Biology, Bioinorganic, Biochemistry
Development of in vivo targeted MRI contrast agents
Fig 4. Schematic of Gd-chelate attached to encapsulated polymer and parameters important for enhanced relaxivity
Fig 5. Cryo TEM reconstruction of the bacteriophage P22 - the protein cage nanoparticle used my lab.
The overall goal of this work is the development of powerful targeted imaging probes for the non-invasive assessment of atherosclerosis biology in order to visualize and predict disease progression and complications and guide or monitor therapy. This work is part of a long-term collaboration with Prof. Michael McConnell (Stanford University, School of Medicine - Cardiology) and Prof. Peter Prevelige (Univ Alabama, Dept Microbiology). The biological state of the vessel wall (e.g., inflammation, angiogenesis) is a major factor in the progression and mortality of cardiovascular diseases including atherosclerosis and aortic aneurysms. Cellular and molecular imaging techniques offer significant promise for the diagnosis of cardiovascular disease because they offer sensitive detection of vascular biological activity in its earliest stages.
Using the ATRP approach described above, we have optimized the imaging capabilities of the bacteriophage P22 capsid and developed a set of atherosclerosis-targeted P22 cages (Fig 4 - The parameters are number of water bound (q), exchange lifetime (Ï„m), and rotational correlation time (Ï„r)). The imaging probe development is centered on the use of the Gd-labeled polymer inside P22 for enhanced T1 relaxivity. These P22-capsid-polymer hybrid materials have the advantage of high loading capacity of Gd-chelates, long rotational correlation times, and only slight decrease in the water exchange rate at the Gd-chelate as compared to the free Gd-chelate. Together these properties result in extremely high T1 relaxivity enhancement (on the order of 650,000 mM-1s-1 per particle at 1.5 T), making them promising MRI contrast agent candidates. The mouse model was injected with (a) Magnevist and (b) Gd chelate attached to polymer encapsulated within the protein cage - at 10x lower [Gd] dose than (a). The yellow arrow indicates the atherosclerotic left carotid artery (right artery is â€˜normalâ€™). This view is a virtual transverse section through the neck of the animal).
In vivo behavior and targeting: We have shown that targeting peptides can be chemically or genetically appended to the exterior of the P22 capsid (and a variety of other protein cage architectures) and function in vivo to direct a substantial portion of the cages to tissue of interest (i.e. atherosclerotic plaque and abdominal aortic aneurysm). These include peptides such as RGD (which target cell-surface integrins) and lyp-1 (which targets activated macrophages) that can be incorporated onto the cage using site selective attachment chemistry or as a peptide insertion into a loop region on the protein surface. My newly funded R01 grant from NIH focuses on developing a superior P22-based nanomaterial with optimized in vitro properties such as high T1 relaxivity, but also investigates the in vivo behavior for tissue specific of these MRI contrast agents in atherosclerosis and abdominal aortic aneurysm animal model systems (Fig 5 - A) Structure of the infectious P22 bacteriophage B) Heterologously produced empty capsid of P22 with schematic representations of surface presentation of targeting ligands and C) Internal polymer with pendant Gd-chelates (up to 40,000 per cage) chemically anchored to the interior surface).
To control the in vivo characteristics we are using techniques of phage genetics to generate libraries of mutant P22 phage that can be used to screen for enhanced circulation time and/or tissue targeting or altered immune response. The mutations that result in desirable pharmacokinetics can easily be integrated with our synthetic efforts using techniques of molecular biology. The P22 phage system ensures that the targeting peptides perform identically in the synthetic P22 and discovery (phage) platforms. Current systems employ targeting peptides discovered in one system and grafted onto another in a poorly characterized manner and in environments that might vary from the peptide presentation on the phage during discovery. Our use of the P22 bacteriophage as a novel in vivo phage display system, which is structurally identical to the engineered (and synthetically manipulated) P22, means that targeting moieties are in precisely the same local environment for both discovery and in vivo use.
Synthesis, Structure, Spectroscopy, Protein Chemistry, NMR, Inorganic, Chemical Biology, Biophysical, Bioinorganic, Biochemistry, Analytical
Enzyme (and other gene product) Encapsulated Nanoreactors
Fig 6. Protein based enzyme encapsulation compartments.
Fig 7. Schematic representation of cargo protein loading inside of the P22 capsid.
A current challenge in bio-mimetic materials chemistry is the development of functional nanomaterials that integrate versatile supramolecular assemblies with the power of enzymatic catalysis as seen in biology (Fig 6 - (A) ethanlamine utilization (Eut) microcompartment. (B) carboxysome microcompartment. (C) T. maritima encapsulin. (D) B. subtilis lumazine synthase. (E) pyruvate dehydrogenase complex. (F) human ferritin. (from Ref )). Such materials would dramatically extend biotechnologyâ€™s range of applications, allowing biocatalysts to be used in contexts very different from their evolved cellular role. In a new class of bio-inspired materials, the directed confinement of enzymes (or other gene products) within protein cage assemblies is a powerful strategy for combining the advantages of the viral architecture with catalytic function and using genetic manipulation as a synthetic tool.
The bacteriophage P22 uses a scaffold protein (SP) to direct the assembly of its coat protein (CP) into icosahedral capsids which are stable to above 85Â°C. Creating a genetic fusion of a desired cargo protein with a modified SP, results in the co-assembly of SP-fusions and CP into a stable icosahedral â€œnano-reactorâ€ in which the cargo is sequestered inside the P22 capsid (Fig 7 - Recombinant coat proteins and scaffold proteins (fused with a cargo gene product) retain the ability to co-assemble to form the icosahedral capsid). These functionalized capsids self-assemble when expressed in E. coli and encapsulate up to 350 copies of the SP-fusion protein within the capsid. We can utilize the tools of molecular biology and genetic engineering to harness the wealth of biological catalysts, packaged (at high local concentration) into P22 capsid that closely mimic the active biological assemblies shown in Fig 7.
We have demonstrated programmed encapsulation of up to 400 copies of fluorescent proteins (GFP and RFP) either singly or co-packaged within a single capsids . We also have a range of enzymes packaged into the capsid of P22 in very high copy number (200-400 enzymes per capsid) and have demonstrated that they retain their activity, although modulated (both the rate and substrate affinity are affected) by the morphological form of the P22 capsid. The concentration of enzyme entrapped inside the capsid is on the order of 5-30 mM (depending on the size of the enzyme). By directing the encapsulation of multiple copies of enzymes, within the confined environment of the P22 capsid interior, we can investigate enzymatic reactions in more â€œcell-likeâ€, crowded environments with physically enforced proximity. The protein cage can be separately optimized as a container that can shield the enzymatic cargo from its environment, enhance stability, modulate enzymatic activity through crowding effects, provide in vivo targeting, or directed assembly into higher order structures.
We are currently working to create synthetic â€œmetabolonsâ€ whereby multiple different enzymes can be incorporated into a single capsid so that the product of the first enzymatic reaction is the substrate for the second enzyme, etc. Small molecule substrates diffuse through the capsid shell while the enzymes are trapped inside like a â€œship-in-a-bottleâ€. Since we have genetically programmed the directed self-assembly of these systems, there is a large scope for the types of enzymes we can utilize. Current projects include the encapsulation of phosphotriesterases (which cleave organophosphates like the nerve agent sarin and pesticides), lactonases (which cleave the N-acyl homoserine lactones that are the quorum sensing molecules for bacterial biofilm formation), hydrogenases (which catalyze the reduction of H+ to form H2), catalases and peroxidases (for scavenging ROS), silicateins (for catalytic hydrolysis of metal ion complexes), as well as a wide range of polypeptides  (including glutathione-like repeats (ECG)n, Fe2O3 nucleating peptides , TiO2 and ZnO nucleating peptides, elastin-like sequences that are thermally responsive, conotoxin peptides, and defensin peptides). The polypeptide encapsulation follows a parallel track to the synthetic polymer approaches described above.
1. S. Kang and T. Douglas:
Science, 2010, 327, 42-43.
2. A. O'Neil, C. R. Reichhardt, P. E. Prevelige and T. Douglas:
Angewandte Chemie, 2011, 50, 7425-7428.
3. C. R. Reichhardt, A. O'Neil, M. Uchida, P. E. Prevelige and T. Douglas:
Chemical Communications, 2011, 47, 6326-6328.
Synthesis, Structure, Protein Chemistry, Mechanism, Inorganic, Chemical Biology, Biophysical, Bioinorganic, Biochemistry
Other Projects in the Douglas Group
a) Long-term collaboration with Prof. Yves Idzerda (Dept of Physics, Montana State University) on magnetic nanoparticles. Understanding the magnetic properties of nanoparticles, encapsulated with the supramolecular architectures of a range of protein cages. This involves synthesis, magnetic characterization (ACMS, Magnetometry), structural characterization (X-ray scattering, PDF), and electronic structure charactrerization (XAS, XMCDS). This work is currently funded through a collaborative grant (NSF-NIRT, CBET).
b) Collaboration with Prof. Allen Harmsen (Dept of Immunology and Infectious Disease, Montana State University)
Introduction of protein cages, such as P22 and the small heat shock protein from Methanococcus jannaschii, induce iBALT (inducible bronchus associated lymphoid tissue) in the lung when administered to mice (and ferrets). This transitory tissue results in complete protection of the animals against lethal pathogens (influenza, SARS, MRSA, respiratory syncytial virus) without the presence of damaging inflammation . We have submitted a grant on this work to support development of these supramolecular architecture-based â€˜vaccinesâ€™.
c) Collaboration with Prof. Robb Cramer (Dept of Immunology and Infectious Disease, Montana State University). We have demonstrated that P22 is rapidly taken up by the growing tip of the hyphae yeast Aspergillus fumigatus. The uptake of P22 can be readily visualized when the capsid is loaded with GFP (or RFP). Alternatively the P22 capsid, when polymerized with polyAEMA provides a very high density of positive charge on the interior and up to 300 copies of a 500bp dsDNA strand can be loaded inside the capsid. Delivery of DNA by P22 is being explored as a potential method for therapeutic delivery of nucleic acids. In addition, encapsulation of small polypeptides known as defensins, which have high antimicrobial activity, within the P22 capsid (via scaffold protein fusion) is being explored for anti-fungal activity.
d) Collaboration with members of the Thermal Biology Institute (MSU) and Prof. John van der Oost (Wageningen University, Netherlands) to identify and use thermal stable enzymes and protein architectures, from extreme environments (such as Yellowstone National Park). Over the last decade my lab has found a large number of extremely thermal stable systems that we now have in hand to exploit for materials applications.
e) Collaboration with Prof. Mande Holford (Department of Chemistry, Hunter College, NY). Encapsulation of omega-conotoxins (small 20-30 AA long peptides derived from the cone shell mollusk) to the interior of the capsid. The conotoxins are â€˜toxinsâ€™ and not easy to produce heterologously. Using our P22 encapsulation approach we can encapsulate up to 400 copies of the peptide in each P22 capsid. The toxins are used therapeutically and their encapsulation within the P22 capsid is being explored for targeted delivery applications.
1. J. A. Wiley, L. E. Richert, S. D. Swain, A. Harmsen, D. L. Barnard, T. D. Randall, M. Jutila, T. Douglas, C. Broomell, M. Young and A. Harmsen:
Plos One, 2009, 4, e7142.