Group Members |
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From Left: Robert Lawrence, Robin Paul, Balakumar Thangaraj, Raimund Fromme, Justin Flory, Alan Lee,
Jason Grayslak, Zhen Gong, Petra Fromme, Ingo Grotjohann, Kimberly Rendek, JayHow Yang, James Zook |
Post-Doctoral Researchers |
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Ph.D. Students |
- James Zook
James Zook is a 2007 graduate from the University of Wisconsin - Eau Claire with a B.S. in Biochemistry and Molecular Biology. He joined the Fromme lab in 2007.
His research centers around the structural characterization of the membrane protein, OEP16. Amino acids are a crucial substrate for chloroplasts due to their ability to express proteins that are encoded within the organelle. This small 16kDa protein is responsible for the passive diffusion of amino acids across the outer membrane of chloroplasts.
Techniques involving this research include Recombinant DNA Stratagies for obtaining large quantities of protein, Circular Dichroism Spectroscopy for the investigation of secondary structure and thermal stability, Light Scattering for determination of multimeric state, and Nuclear Magnetic Resonance for tertiary structure conformation. NMR has been an emerging technique in the high resolution structure determination of proteins for proteins that strongly resist crystallization for diffraction studies.
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- Robert Lawrence
Robert Lawrence is a 2005 graduate from the University of Utah with a B.S. in Chemistry.
The chloroplast ATP synthase is a large multisubunit complex that is rooted in the thylakoid membrane by subunits a, b, and a ring of c subunits. The ring of c subunits provides the rotational force that drives ATP synthesis in photosynthesis. This occurs as individual protons bind to individual c subunits, which causes a rotational shift of the c-subunit ring as protons are translocated from the lumen to the stroma. It is known that the number of c-subunits per ring varies among organisms, which in turn results in a variation of the corresponding coupling ratio (H+ transported per ATP synthesized). The cause of this c-subunit variable stoichiometry is not well understood, and his research is devoted to investigating factors that may be of influence.
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In order to accomplish this, Robert has worked on developing a recombinant expression system that allows the chloroplast ATP synthase c-subunit to be produced in E. coli. Results have shown that this system is able to produce large amounts of purified, folded recombinant c-subunit, which can be used for further applications. Currently, he is exploring ways to reconstitute the subunits into a ring. This will enable a variety of structural investigations into c-subunit ring stoichiometry that have not been previously possible. The developed expression system may also be applied to other eukaryotic membrane proteins, which are notoriously difficult to produce using recombinant bacterial systems.
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- Kimberly Rendek
Kimberly Rendek is a 2008 graduate from Arizona State University with a B.S. in Biology.
Kim Rendek is a third year biochemistry Ph.D. student at Arizona State University focusing mainly on bioenergy, biochemistry, molecular biology, and X-ray crystallography. Her primary work includes the design of an artificial oxygen-evolving complex (aOEC) that will be incorporated into solar fuel cells as part of an Energy Frontier Research Center (EFRC) project. The need for a renewable and sustainable energy source is crucial, and this work adopts a bioinspired approach, emulating the water-slitting capabilities of Photosystem II, nature’s best kept secret. In cyanobacteria and plants, due to the instability of Photosystem II’s primary electron donor, the highly oxidizing P680+, Photosystem II’s core protein, D1, is degraded every half hour in the presence of sunlight. Thus, it is necessary for the aOEC to be contained in a stable framework, without the presence of P680+. The stable framework used for this project is a three-dimensional DNA tetrahedron, which is used to encapsulate synthetic peptide sequences based on the high-resolution X-ray structure of cyanobacterial Photosystem II, which reveals the amino acids that provide ligations to the manganese cluster (4MnCaCl). As a preliminary study, the DNA tetrahedron itself has been characterized using X-ray crystallography, which will provide precise atomic positions that will be used to directly attach synthetic peptide sequences. The overall goal is to successfully design, assemble, and characterize the aOEC through the use of X-ray crystallography, electrochemistry, and electron paramagnetic resonance (EPR).
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Additionally, Kim works on the recombinant expression of human hemoglobin in Synechocystis sp. PCC 6803, a type of cyanobacteria. Cyanobacteria produced the oxygen present in our atmosphere 2.5 billion years ago. Synechocystis specifically is a readily transformable organism and has many characteristics that make it an ideal candidate to host the human hemoglobin protein. Synechocystis synthesizes large amounts of Protoporphyrin IX, a precursor for both heme and chlorophyll biosynthesis. Considering heme is a cofactor in hemoglobin, it is probable that the hemoglobin protein will fully assemble in the cell. The relevance in this work is that currently, blood used for transfusions is obtained solely from expired blood samples and is thus not renewable or safe, as bloodborne diseases can be transmitted. By recombinantly expressing human hemoglobin in a low-cost, light-driven process, we can provide a constantly renewable and safe blood constituent.
Lastly, Kim also works on characterizing two de novo designed four-helix bundle proteins, S824 and S836, capable of binding heme in their cores. This project is a collaboration with Professor Michael Hecht and Dr. Izhack Cherny at Princeton University. The NMR structures of these proteins have been solved, but without the heme bound in the center. Kim’s role in the project is to find ideal crystallization conditions for S-824 and S-836 and eventually solve the X-ray structure of these proteins with heme bound in the core. This work has relevance in drug delivery and design, and electrochemistry.
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- Jayhow Yang
JayHow Yang is a third year Ph.D. student in Biochemistry at Arizona State University, joining the Fromme Research Group in January 2009. Prior to beginning his graduate studies at ASU, in July 2001, he received a B.S. in Forestry at the Chinese Cultural University in Taipei, Taiwan. He continued his studies at Griffith University in Queensland, Australia, receiving a M.S. degree with honors in July 2004, completing his thesis, “Isolation and Identification of Microorganisms Associated with the Cyanobacterium Cylindrospermosis raskiborsii”. Upon fulfillment of his M.S. degree, JayHow gained experience isolating and characterizing chlorophyll pigments and inner and outer membrane components in chloroplasts, working under the supervision of Dr. Chi-Ming Yang at the Research Center for Biodiversity at the Academia Sinica, Taiwan.
JayHow’s Ph.D. studies focus on the isolation and purification of the amino acid transporter OEP16 from Pisum sativum (pea plant) and Spinacia oleracea (spinach). OEP16 is a small, 16 kDa membrane-integral amino acid transporter protein for which the structure has yet to be revealed. JayHow uses analytical centrifugation (sucrose gradients) to isolate the inner and outer membrane constituents of the pea plant and is able to identify OEP16 through the use of SDS-PAGE and immunoblotting techniques. Following purification, crystals of OEP16 have been produced. Further X-ray diffraction studies will be attempted in order to solve the structure of OEP16, which would contribute greatly to the field of membrane transport proteins, notoriously challenging to purify and characterize.
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Another focus of JayHow’s research is the isolation, purification, and subsequent crystallization of the intact ATP synthase from spinach. ATP synthase is an enzyme responsible for producing ATP from ADP and inorganic phosphate using the energy obtained from an electrochemical gradient occurring across the membrane separating the lumen and the stroma in the chloroplast. ATP synthase has a prominent role in the photosynthetic electron transport chain. Currently, no structure of the intact ATP synthase exists. Elucidating the structure of the intact ATP synthase would be of importance for the bioenergy and photosynthesis community.
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- Alan Lee
Alan Lee's research is on the structure determination of fusion protein consist of cholera toxin B-subunit (CTB) and membrane proximal region (MPR) of gp41 in the human immunodeciciency virus (HIV). CTB-MPR is a candidate AIDS vaccine that has shown to elicit HIV-transcytosis blocking antibodies. To further improve the design of the vaccine, it is important to determine the three-dimensional structure of the CTB-MPR.
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- Justin Flory
Justin Flory is a 2002 graduate from the Sonoma State University with a B.S. in physics. He is currently in the Biological Design program here at ASU.
Justin is deeply concerned about climate change and the impact of human development on the environment. Human society depends on the environment for many services like energy, clean air and water. Justin is a part of the Center for Bio-Inspired Solar Fuel Production sponsored by the Department of Energy, which is developing low cost methods to convert solar energy into hydrogen gas as a replacement for fossil fuels.
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- Zhen Gong
The envelope glycoprotein (Env) complexes of the human immunodeficiency viruses (HIV) mediate viral entry and are a target for neutralizing antibodies. Knowledge of the trimeric Env structure is essential for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. The Env complex is initially assembled from three copies of the precursor polypeptide gp160, which are cleaved to yield a final mature trimer of heterodimers of the gp120 and gp41 subunits. Zhen Gong's project is collaboration with Dr. Mor and his Ph.D. student Sarah Kessans from life science of ASU. The first aim is to solve the structure of gp41 by X-ray crystallography or NMR. Then they will design the vaccines of HIV according to its structure. Currently they are exploring different expression strategies for various gp41 constructs in E.coli and plants.
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Zhen’s second project is related to the work on the structures of supercomplexes of the Photosystems with their peripheral antenna complexes, with special focus on Photosystem I. She is trying to solve the structures of the supercomplexes and the individual building blocks. Currently she is investigating the structure of phycoerythrin (PE) from marine cyanobacteria. She also carries out studies on restructuring of the photosynthetic light-harvesting antennae, or phycobilisomes (PBS), which are responsible for providing light energy to both Photosystem I and II and investigate the change of the Photosystem structures during chromatic adaptation. She grows the cyanobacteria under different wavelengths of light and analyze the protein ratio of phycoerythrin (PE) and phycocyanin (PC) in relationship to the Photosystem I (and II) supercomplexes.
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- Robin Paul
Robin Paul’s research project involves the study of change in expression of phycobilliproteins with change in light conditions, a phenomenon known as chromatic adaptation in a new strain of filamentous cyanobacteria. This involves techniques like electron microscopy which enables study of this phenomenon at the cellular level and protein crystallography at the molecular level. He is currently involved in the crystallization of phycoerythrin (a phycobilliprotein) which may give new insights into the assembly of the phycobillisomes. Another goal of his project is to isolate photosystems from this strain and solving its structure using X-ray crystallography.
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- Mark Hunter
Mark's Photo
Mark Hunter is a 2006 graduate from Wilkes University with a B.A. in Chemistry.
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