Harvesting the redox power from photosynthetic bacterial reaction centers
We are working together with Don Seo’s team in the School of Molecular Sciences (https://sms.asu.edu/dong_seo) to learn how to effectively interface the light-induced redox potentials generated by photosynthetic reaction centers from purple bacteria with novel nano and meso-structured electrode materials. Interfacing redox active proteins with electrode materials is a important goal. This would open the door to a wide range of applications involving the exchange of energy and information between biological and electronic systems. Photosynthetic reaction centers are an ideal model with which to explore such an interface as their redox reactions are light driven, as opposed to requiring a substrate, allowing multiple turnovers and adding an additional level of control.
Orange Carotenoid Binding Protein (OCP)
Photosynthesis is a fascinating molecular process that captures light and transforms it in chemically available energy to sustain life. Our work focuses on trying to understand the underlying mechanisms, and how the components of this system work, both for fundamental research, as well as for applications. In our lab we are working on designing and implementingartificial photosynthetic complexes by using the power of DNA nanostructures to precisely assemble organic dyes to harvest light, and reaction center proteins to convert light into charged states that can drive enzymatic reactions. To further mimic the natural system, we aim at inserting a control element in these artificial networks. The inspiration comes from cyanobacteria: in these microorganisms, the Orange Carotenoid Binding Protein (OCP) regulates the transfer of excitation energy in the antenna system by acting as an energy quencher when the antenna is over-excited by light. We are currently working to assemble the OCP within a DNA-based artificial light harvesting system, to act as a regulatory element that can selectively quench the fluorescence of dyes when strategically placed. As a proof of principle we have assembled a antenna system on the dyes Cy3 and Cy5 attached to a 3-way DNA junction, and have incorporated OCP as a control element. We have been able to demonstrate the regulation of energy transfer between the Cy3 and Cy5 dyes as a function of light intensity. This proof of concept moves us a step closer to the production of controllable systems for harvesting and utilizing the energy of light.
Role of Protein Dynamics in Photosynthetic Electron Transfer
We have a fundamental research program focused on the study of electron transfer in bacterial photosynthetic reaction centers on various timescales. This work utilizes ASU’s ultrafast laser facility to follow these electron transfer reactions on the femtosecond to microsecond timescale. This work in performed in close collaboration with the laboratory of Dmitry Matyushov who has developed computational approaches for analyzing dynamics in non-ergotic systems. It has become clear that the kinetics of electron transfer in these reaction centers is directly limited by protein motion. Much of our effort in this area involves comparing the computational models with the measured behavior and trying to modify specific reaction parameters that affect the models such as free energy and protein flexibility.
Photoprotection Mechanism in Bacterial Photosynthetic Reaction Centers
Photosynthetic organisms collect light energy and convert it to electrochemical potential primarily by using light harvesting complexes and reaction centers (RCs). Light harvesting complexes contain an array of chlorophylls and carotenoids embedded in protein scaffolds. The RC protein complex contains three sub-domains namely, L, M, and H which support a number of redox cofactors including a special pair (P, bacteriochlorophyll dimer), two bacteriochlorophyll monomers, two bacteriophyeophytin and two quinones and a carotenoid. Upon light excitation of P, the redox cofactors take part in an electron transfer chain reaction to develop potential gradient across the membrane. Carotenoid in both light harvesting and RC protein complexes play a dual role in both light harvesting via energy transfer to bacteriochlorophyll and photoprotection through the dissipation of excess energy. Several different mechanisms have been proposed to explain how carotenoids are involved in energy dissipation in various photosynthetic systems. In bacterial reaction centers, it is thought that triplet-triplet energy transfer from the triplet excited state of P to the carotenoid is the key step in preventing the generation of reactive singlet oxygen species. However, the exact mechanistic pathways and the kinetics for the triplet energy transfer in RCs are still elusive. We are using pump-probe transient absorption spectroscopy over a wide temporal range from femtoseconds to sub-milliseconds to probe the energy transfer dynamics of photoprotection in the RCs of Rhodobacter Sphaeroides.
Immunosignature assays and peptide arrays
An immunosignature assay is a measure of the profile of antibodies circulating in the blood. The measurement requires only microliters of blood, diluted ~1000 fold and applied to a 0.5cm2 array containing ~125,000 peptides that have been synthesized in situ. Because the measurement is the overall antibody profile, it is disease agnostic; the same physical array, indeed the same drop of blood, can be used to identify any condition that affects the profile of circulating antibodies. This is true for humans or animals. The technological concept is similar to a standard ELISA (enzyme-linked immunoassay) commonly used in serum-based diagnostics. However, while an ELISA would provide only a single reading, the immunosignature results in a 125,000 component signature for each sample assayed. For each disease, a small subset (50-5000) of the peptides in the array comprise a distinctive signature for that disease. We have currently shown promising results for detection of more than 30 diseases and a company, HealthTell, has been spun out around this technology (www.healthtell.com ).
There are many aspects to the current research in this area. We are developing the peptide arrays themselves to do a better job of distinguishing between diseases and detecting them early. We are trying to understand the thermodynamics and kinetics of antibody binding to the arrays and how that affects the information content of the meausurements. We are considering other types of measuring modalities (currently we measure the antibodies using scanning lasers, but direct electronic detection would allow the development of inexpensive, portable devices). We are developing new approaches to analyzing the data both in terms of the algorithms themselves (e.g., machine learning) and in terms of the kinds of samples and measurements that should go into the analysis. Finally, we continue to explore the application of the approach to new diseases, to vaccine production and to epidemiological surveys both locally and internationally.
The peptide arrays themselves have many other potential applications beyond the immunosignature technology. The arrays are synthesized directly on silicon wafers using the same types of instruments involved in electronics fabrication. The peptide libraries made have been used for a variety of purposes including selecting peptide ligands, selecting potential antimicrobial sequences and finding peptides that specifically modify enzymatic activity. Our Peptide Array Facility (peptidearraycore.com) is open for outside users and we have collaborations with numerous groups. The facility is now part of ASU’s Nanotechnology Collaborative Infrastructure Southwest (http://ncisouthwest.org/index.php/peptide-array-core/ ) and provides arrays applications and training nationally.