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Stefan Franzen

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Grants

Date: 01/01/21 - 12/31/23
Amount: $474,000.00
Funding Agencies: National Science Foundation (NSF)

The protein structure-function correlation has been built from decades of study of primarily monofunctional systems, but whether (and how) structure uniquely defines function is a significantly more complex subject to address. This complexity is easily seen within the heme protein superfamily, where functions as diverse as O2-binding, oxygenase, oxidase, peroxygenase, and electron-transfer all occur at active sites that often contain a surprising number of similarities, both structural and mechanistic, but where the protein environment exquisitely controls the chemical reactivity of the heme group to ensure maximum intended function with a minimum of unintended cross reactivity. Understanding how Nature controls for the selectivity of function (‘activity differentiation’) across the heme protein superfamily has been a longstanding question that penetrates to the core of the protein structure-function correlation. Yet despite decades of study, fully addressing this question remains a challenge given the inherent structural bias towards a single function that arises when studying purely monofunctional heme systems. As a model for probing the protein structure-function correlation in the hemoprotein superfamily, our work has focused on the enzyme dehaloperoxidase (DHP), the coelomic hemoglobin from the marine worm Amphitrite ornata. Beyond its oxygen transport function, DHP has been shown to possess four additional activities (peroxidase, peroxygenase, oxidase and oxygenase), all of which employ a common Fe(IV)-oxo intermediate. Remarkably, in a paradigm shift from the traditional protein structure-function correlation, we have found that the substrate itself, and not the protein structure, plays a pivotal role in determining which enzyme activity is performed by DHP. Termed the Substrate Directed EnzymatiC Activity paRadigm (“SiDECAR”), the choice of DHP activity “encoded” by the substrate enables us to interrogate in detail how Nature controls for activity differentiation across the heme protein superfamily: simply by changing the substrate, we can investigate the protein structure-function properties specific to four different activities using just a single enzyme, allowing us to pose questions related to how proteins control for electron and/or proton transfer, how specific amino-acid/substrate interactions influence oxidation mechanisms, and the role(s) of both conserved and non-conserved residues that enable specific discrete functions within the heme protein families. Thus, DHP provides an unprecedented platform for deeply probing questions relating protein structure to the tuning of an enzyme’s activity (without bias from studying a monofunctional protein), and will be explored in the following two Specific Aims: Specific Aim 1: To elucidate the mechanism of oxyferrous DHP activation by neutron diffraction studies. Hypothesis 1: Substrate binding to oxyferrous DHP (hemoglobin active) leads to a tautomerization of the distal histidine that destabilizes the bound molecular oxygen toward reaction with H2O2, leading to Compound II (enzyme active/catalytically relevant) via a putative ferrous-hydroperoxide intermediate. Objective 1: The objective of this aim is to identify the key protein residues (e.g., the distal histidine) that are involved in DHP activation from the oxyferrous state in the presence of substrate, and to see if these residues change as a function of the “SiDECAR” activity differentiation in DHP. Specific Aim 2: To perform DHP mechanistic studies using advanced X-ray structural methods. Hypothesis 2: A combination of structural features in DHP (distal His55, hydrophobic cavity) and substrate properties (pKa, binding orientation) lead to the “SiDECAR” activity differentiation in DHP. Objective 2: We will probe the peroxygenase, oxygenase and oxidase mechanisms of DHP using time-resolved X-ray crystallography, the multiple structures serially from one crystal (MSOX) method, and EXAFS to explore intermediates or initiate time-resolved studies relevant to catalytic turnover. The

Date: 09/01/16 - 12/31/20
Amount: $524,195.00
Funding Agencies: National Science Foundation (NSF)

The protein known as dehaloperoxidase-hemoglobin (DHP) has provided us with a recent example of a multi-functional protein that challenges many of the assumptions behind the structure-function correlation. Of course, structure is related to function, but the question is whether structure uniquely defines function. DHP appears to suggest that structure and function can be tuned by subtle conformational control of the conformation of a few amino acids near a metal binding site (Fe in this case) to provide functions in oxygen transport and the enzymatic dehalogenation of halophenols, but more also recently established peroxygenase, oxidase, and oxygenase activities. The focus of the proposal will be a combined structural, spectroscopic and mechanistic (kinetics) study that will elucidate the specific set of substrate/inhibitor interactions and dynamics changes for each of the four known functions of DHP. The knowledge gained will contribute to the elaboration of the structural features and other determinants that impart specific discrete functions to heme proteins (and more generally to all proteins).

Date: 09/01/15 - 8/31/20
Amount: $500,000.00
Funding Agencies: National Science Foundation (NSF)

The concept of finding new semi-conductor materials capable of surface plasmon resonance in a portion of the electromagnetic spectrum that is amenble to measurement is now established. In fact, the idea of using conducting metal oxides for this purpose was first suggested by the PI in 2002 and first demonstrated the Franzen and Maria groups in 2006. Since that time the interest in this area both in the community and in our research groups has heightened dramatically. We now realize that applications include, not only sensing, but also potentially new means of information storage and switching. Conducting metal oxides are only a first step in the direction of new semi-conductor compatible materials. During our current funding period we have extended this idea to the realm of III-V semi-conductors themselves. We constructed three different mid-IR plasmonic spectrometers and demonstrated sharp SPR in high mobility CdO thin films. This proposal develops this breakthrough for applications in the mid-infrared region of the electromagnetic spectrum.

Date: 08/15/11 - 7/31/16
Amount: $514,000.00
Funding Agencies: National Science Foundation (NSF)

Optical studies of conducting thin film materials provide a wealth of information that can be used to design new materials with novel optical properties. Research in the PIs group has recently shown surface plasmon resonance (SPR) can be observed in indium tin oxide (ITO) thin films in a manner that is analogous to the SPR effect on Au and Ag, which is widely used in sensor applications.

Date: 09/01/10 - 8/31/14
Amount: $434,722.00
Funding Agencies: US Army - Army Research Office

Chlorinated phenols are persistent chemical pollutants that upon exposure have been shown to give rise to both acute and long-term health risks. Many chlorinated phenols reside in soils following treatment with pesticides or defoliants, or are produced as byproducts of paper production or incineration, where they present a danger if they enter the food supply even at the parts per trillion level. The proposed research herein addresses the urgent need to incorporate biological strategies into environmental restoration efforts (bioremediation) that focus on the catalytic degradation of chlorinated phenols such as 2,4-dichlorphenol and 2,4,5-trichlorophenoxyacetic acid (a component of Agent Orange). By focusing on enzymes as bioremediation catalysts, the proposed effort may lead to the development of novel proteins capable of the catalytic degradation of the aforementioned chlorinated phenols, thereby minimizing their deleterious effects on the environment. The kinetics, spectroscopy, structure, and engineering of three such systems will be explored in detail: soybean seed hull peroxidase (SBP), dehaloperoxidase (DHP) from the marine annelid Amphitrite ornata, and the hydroxylase from C. necator. Employing natural products from soybeans or living bacteria to degrade these wastes has enormous potential to positively impact the environment, and may lead to the development of a new bioremediation industry.

Date: 07/01/11 - 6/30/12
Amount: $4,000.00
Funding Agencies: NCSU Faculty Research & Professional Development Fund

The delivery of oligonucleotides to cells is of vital interest in all aspects of molecular biology and biomedical research. This long-standing problem has been approached using a variety of chemistries to condense and package nucleic acids using cationic polymers, dendrimers, liposomes, and nanoparticles for drug delivery applications. The use of viruses to delivery genetic material continues to be an alternative that has great potential. Viruses are uniquely adapted to the packaging and transport of genetic material. However, host viruses such as the adenovirus poses severe health concerns that has led to an extensive effort to genetically modify such viruses. It is still not clear that host viruses will ever be completely safe. In recent years, the PI has focused on an alternative to these technologies using the plant virus, Red clover necrotic mosaic virus (RCNMV). The fundamental problems associated with the use of RCNMV drive the need to expand the research focus to the Turnip yellow mosaic virus (TYMV). This proposal represents a new direction that will lead to delivery of ribonucleic acids (RNAs) to cells using TYMV as a platform technology that promises to be superior to the RCNMV for this crucial application. In order to make this transition and to propel the research group in a new direction, the PI is requesting support under the FRDP mechanism.

Date: 04/01/07 - 9/30/10
Amount: $157,509.00
Funding Agencies: National Science Foundation (NSF)

This proposal describes a summer undergraduate student program at Adam Mickiewicz University (AMU) and the Institute of Bioorganic Chemistry of the Polish National Academy of Sciences (IBCh) in Poznañ, Poland. The program involves an educational and undergraduate research component to research projects that involves collaborative research on problems of general interest for RNA structure and function. The theme of the research is the study of structures of oligonucleotides that are involved in viral coat protein assembly using methods that include modified fluorescent oligonucleotides. The PI and co-PI have recently embarked on program of research that involves the development of novel plant viral nanotechnology. We have collaborated with the Polish research groups of Prof. Bohdan Skalski (AMU) and Prof. Ryszard Adamiak (IBCh) for the past six years. This collaboration has included joint publications, patents, reciprocal visits to laboratories, seminars collaborators universities, short courses at AMU visits by graduate students and a summer undergraduate research project initiated in 2006. Over the course of the past 6 years the PI, co-PI and Polish collaborators have developed the undergraduate summer research program described here. The program will benefit from synergistic projects that have common features. We have found a compromise the benefits the research projects in collaborators laboratories while providing a coherent training for students.

Date: 08/01/08 - 7/31/10
Amount: $239,426.00
Funding Agencies: NC Biotechnology Center

The development of targeted drug delivery systems requires a rapid and efficient method for determining the biodistribution of the targeting agent. We have recently demonstrated that PVN targeting agent is capable of at least three modes of modification with application to cell targeting. The modes of modification are 1.) encapsidation of cargo, 2.) infusion of drugs and biosensor and 3.) surface modification with targeting molecules. We have performed in vitro studies that demonstrate both the targeting and delivery potential of this platform technology. Those studies have led to the formation of Nanovector, Inc. a formation stage company that has targeted delivery of chemotherapy agents to cancer cells. In the process of considering the development of the technology for therapeutic agents we have also realized the potential for delivery of biosensors, oligonucleotides for gene splicing and gene silencing by RNA interference as applications for PVN technology. The hurdle to commercialization is similar to that faced by a number of platform technologies. How do we ensure that the in vivo targeting is specific and free from adverse side reactions? This is a complicated question since it involves both immunogenicity and cell specificity of the chosen mode of targeting. While antibodies have tremendous specificity for targeting, their ability to carry cargo is limited to attached molecules. The PVN approach allows a significant cargo to be carried into a cell and released. Based on these considerations we have designed a new type of research tool for the observation of targeting in vivo.

Date: 09/30/09 - 3/30/10
Amount: $11,736.00
Funding Agencies: National Institutes of Health (NIH)

The project investigates a method for transporting anti-cancer drugs into cancer cells without affecting surrounding healthy cells. We use a small protein shell, which can be armed with small signals that recognize cancer cells because of their tendency to produce a large number of specific proteins on the cell surface and can be loaded with known anti-cancer drugs to increase their effectiveness because of specific targeting and delayed release inside the cancer cell. The protein shell is a plant virus that naturally has the desirable release properties for cancer treatment, but cannot infect animals or humans.

Date: 03/19/07 - 3/18/10
Amount: $332,913.00
Funding Agencies: US Army - Army Research Office

The project addresses the need to develop new biological approaches to the remediation of the environment. We propose to study the first hemoglobin that has evolved to adopt an additional peroxidase function. The hemoglobin was first isolated from the marine worm (terebellid polychaete) Amphitrite ornata, but has been cloned and expressed in E. coli bacteria. In addition to the new function the enzyme dehaloperoxidase (which is another name for the hemoglobin) has an internal binding site for the substrate. This is an unusual feature in heme proteins. There is no other known hemoglobin or peroxidase that has an internal binding site. Thus, the dehaloperoxidase (DHP) from A. ornata is a unique enzyme. The enzyme DHP is relevant for environmental remediation since chloro- and bromophenols are known substrates for the enzyme. Bromophenols are naturally occurring toxins and chlorophenols are pollutants that are introduced into the environment as herbicides, cleaning agents and from bleaching in the pulp and paper industry. The fact that a worm has adapted to degrade these compounds using a hemoglobin that evolved to assume a second function presents an interesting possible future application of hemoglobins in general. Thus, our research is directed towards understanding bioremediation by hemoglobin dehaloperoxidases with a view of a future research on directed evolution and development of proteins that may have application in blood for rapid detoxification.


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