Directed Evolution of Bacteriorhodopsin for Use in Optical Devices
Field of Interest:
The research project described in this proposal incorporates techniques and skills that are commonly used in chemistry and molecular biology laboratories. While the primary concentration of my undergraduate degree is in chemistry, I have also chosen to major in molecular and cell biology. This coursework, in conjunction with a strong research background in protein biochemistry, have enabled me to contribute to the success of several research projects. The goals described for this proposal require that I use both semi-random mutagenesis and spectroscopy to create and analyze protein variants for select photochemical properties, respectively.
Upon
graduating from the
Goal of Project:
The specific aim of this project is to optimize the protein bacteriorhodopsin (BR) for use in device applications by genetically altering the bacterio-opsin (bop) gene via semi-random mutagenesis. Bacteriorhodopsin is a membrane bound protein consisting of seven transmembrane helices. The protein is expressed by the halophilic archaeon H. salinarium under low oxygen concentrations and high light. Upon light excitation, BR pumps a proton from the cytoplasmic face of the membrane to the extracellular matrix. The resulting proton gradient drives intracellular ATPase to convert ADP and inorganic phosphate (Pi) to ATP. The protein absorbs light via a retinal molecule that is covalently bound to the protein through a protonated Schiff base linkage. Upon light excitation, the chromophore isomerizes from its all-trans resting state, bR, to 13-cis. This begins the photocycle consisting of the intermediates K, L, M, N, and O. A branched photocycle occurs through absorption of a second photon of light while the protein is in the O state. The branched photocycle is defined by the P state (9-cis), which eventually thermally decays into the Q state (hydrolysis of the chromophore, which remains in the protein).3
The branched photocycle is of particular interest because it has a lifetime of 5-20 years and a thermal isomerization barrier (9-cis back to all-trans) of approximately 45kJ/mol. This is approximately the energy required to denature the protein.1 A long Q state lifetime makes the Q-state desirable for use in holographic and volumetric optical memories. By setting both the P and Q states as binary bit 1 and the other intermediates, including the resting state, as binary bit 0 data can be written, read, and erased from the protein.
One of the primary ways to improve the access to the branched photocycle is to lengthen the lifetime of the O state. Protein variants that disrupt the deprotonation of D85 will most likely result in an increase in O production.1 It should be noted that mutants with long P states are undesirable for device applications because this state consists of an equilibrium of two components (P445 and P525). This equilibrium of states may result in loss of data due to unwanted photochemistry.2 Thus, it is important that the Q state is produced efficiently for use in long term data storage devices.
For this project I will be targeting the residues F208, R82, and D85. Semi-random mutagenesis will be used to produce a library of bop variants with mutations in these targeted regions. Presently, F208N is the protein variant that produces the greatest Q state yield. Furthermore, F208N is shown to enhance the lifetime of the O state. Since F208N has been shown to form a significant amount of Q state, other variants in this region may also have improved Q state kinetics.
The regions of R82 and D85 are also thought to contribute to the formation of the Q state. The protonation of D85 is responsible for lowering the transition energy from all-trans to 9-cis retinal, while raising the transition energy of the reverse reaction. Thus, increasing the favorability of branched photochemistry.5 Replacing D85 (low pKa R group value) with another amino acid will result in varied protonation states for this residue. The change in protonation states may stabilize the all-trans to 9-cis photochemistry. During the photocycle, the transformation of the O state back to the resting state is linked to the release of a proton from D85.4 Therefore, if the release of a proton from D85 can be disrupted, the lifetime of the O state will be extended and the chromophore will be converted to the branched photocycle with greater efficiency. Replacing the positively charged arginine with a neutral residue will also disrupt proton release, relative to wild type.6 Together, mutations in the regions of both R82 and D85 should slow the conversion of O to the resting state, ultimately increasing the Q state yield.
The scope of this project will include, but is not limited to, randomly mutating residues F208 and R82/D85. The protein variants will be grown in H. salinarum and isolated as purple membrane patches. The purple membrane suspensions will be characterized spectroscopically for the formation of the Q state. Following data analysis, the protein variants will be sequenced and characterized in thin films and volumetric memories.
Methods:
The strand overlap extension method will be used to insert mutations into the regions of interest.4 Two primers will be ordered for each region, one internal (5’à3’) primer containing a 75% doping frequency for the residue(s) of interest, and another 15mer internal (3’à5’) primer that will overlap with the doped primer upstream of the region of interest. Two PCR reactions will be performed simultaneously with these primers. The first reaction will amplify the template DNA with the forward primer and the 15mer internal reverse primer, creating the 5’ half of the gene. The second PCR reaction will amplify the template DNA with the doped internal forward primer and a reverse primer, creating the 3’ half of the gene. The products of PCR 1 and 2 will be digested with the enzyme dpn1, which cleaves the parental, methylated DNA. A third PCR reaction will then be performed using the products of PCR 1 and PCR 2 as a template to create a semi-random construct of the bop gene. Since the doped forward and the 15mer reverse primers overlap each other, PCR 1 and PCR 2 will serve as templates for each other. This reaction will yield a final PCR product consisting of bop gene variants that are localized to the residue(s) of interest.
The PCR fragments and the pBA1 expression plasmid are then digested with the restriction endonucleases NheI and SacI. The plasmid, pBA1, contains all the elements necessary for replication in E. coli, an ampicillin resistance gene, and contains restriction sites (NheI/SacI) flanking the bop gene. Because the restriction digests do not work with 100% efficiency, a gel extraction will be performed to isolate the DNA of interest. This reduces the percentage of wild type BR that is transformed into H. salinarum. The plasmid and the gene containing complementary sticky ends will then be ligated together, transformed into E. coli, and plated on media containing ampicillin. Ampicillin selection will only allow recombinant plasmids with the ampicillin resistance gene to grow. Select colonies are then grown in liquid media, and the plasmid DNA is purified using the QIAprep Spin Miniprep Kit. The plasmid DNA is then sequenced by the UConn DNA Biotechnology Facility to confirm the presence of mutations in the targeted regions of interest.
All of the mutant colonies growing on ampicillin media will then be pooled together subsequently isolated using the QIAprep Spin Miniprep Kit. The variant pools of plasmid DNA are then transformed into H. salinarum. Through homologous recombination, the bop gene will be inserted into the H. salinarum genome. The resulting transformants are plated on SR+Mev plates and after 7-9 days will be pooled and transferred to plates containing 5-FOA serving as a second method of selection. The resulting colonies will then be grown in 25 mL flasks in batches of 96. Once the cells have grown to density, 200µL of each variant will be stored at -80oC for future studies. The protein will then be isolated and purified through centrifugation and placed in a 96 well plate. The 96-well plate of protein will then be irradiated with high intensity red light for 30 minutes to 3 hours. Red light illumination causes the protein to shift to the branched photocycle. A UV-Vis spectrometer will be used to take a spectrum of absorbance versus wavelength over a large range of wavelengths (approximately 250-750 nm) to observe the formation of the Q-state (lmax=390nm). Once a desirable mutant is discovered the DNA will be prepared for sequencing from freeze-dried cell. If a desirable mutant is found, it will then be characterized in thin films and three-dimensional optical memories.
All of the necessary equipment is provided by Dr. Birge, including the chemicals and protocols required to perform the above procedures. These procedures are detailed in the literature and in the Birge lab manuals. This project will be finished in time for me to write a thesis to be submitted by the end of the spring 2007 semester.
References:
(1) Birge, R. R.;
Gillespie, N. B.; Izaguirre, E. W.; Kusnetzow, A.; Lawrence, A. F.; Singh, D.;
Song, Q. W.; Schmidt, E.; Stuart, J. A.; Seetharaman, S.; Wise, K. J.;
J. Phys. Chem. B. ; (Feature
Article); 1999; 103(49);
10746-10766.
(2) Gillespie, N. B.; Wise, K. J.; Ren, L.; Stuart, J. A.;
Marcy, D. L.; Hillebrecht, J.; Li, Q.; Ramos, L.; Jordan, K.; Fyvie, S.; Birge,
R. R.;
J. Phys. Chem. B. ; (Article); 2002; 106(51);
13352-13361.
(3) Arnold,
Francis H., Georgiou, George. Directed Evolution Library Creation Methods and Protocols. Humana Press:
(4) Popp, A, Wolperdinger, M, Hampp, N,
Bruchle, C, Oesterhelt, D
Photochemical conversion of the
O-intermediate to 9-cis-retinal- containing products in bacteriorhodopsin
films. Biophys. J. 1993 65: 1449-1459
(5) Tallent, Jack R., Stuart, Jeffrey A., Song, Q. Wang, Schmidt, Edward J., Martin, Charles H., Birge, Robert R. Photochemistry in Dried Polymer Films Incorporating the Deionized Blue Membrane Form of Bacteriorhodopsin. Biophys. J. 1998 75: 1619-1634
(6) Govindjee, R, Misra, S, Balashov,
SP, Ebrey, TG, Crouch, RK, Menick, DR.
Arginine-82 regulates the pKa of the
group responsible for the light- driven proton release in
bacteriorhodopsin. Biophys. J. 1996 71: 1011-1023
Spring 2006
Standard Plan of Study University Scholar Plan of Study
Chem 264 Physical Chemistry (4) Chem 264 Physical Chemistry (4)
Chem 265W,C Physical Chemistry Lab (2) Chem 265W,C Physical Chemistry Lab (2)
Chem 232 Analytical Chemistry (4) Chem 232 Analytical Chemistry (4)
Chem 296 Undergraduate Research (3) Chem 296 Undergraduate Research (3)
MCB 209 Struct/Function of Biological Chem 393 Scientific Programming (3)
Macromolecules (3) MCB 313 Struct/Function of Biological
Macromolecules (3)
Prior to the beginning of the University Scholar plans of study I will have finished the 24 required credits for a degree in MCB, however I still need to finish the requirements for the chemistry major. To work toward my chemistry major I will be taking Chem 264, 265WC, and 232. In addition to these required courses I plan to take undergraduate research and two graduate courses, one course in scientific programming (Chem 393) and another course in the role of biological macromolecules (MCB 313). MCB 313 is also offered as an undergraduate course, but by being a University Scholar I will be able to further my knowledge by taking the graduate level course. Both courses will help me with my University Scholar project, Chem 393 will give me experience using scientific programs including various programs and methods used to visualize chemical interaction. This will be important so that I can interpret my project results by determining theoretical residue interactions. MCB 313 will be useful because the structural and functional changes resulting from amino acid substitutions on a protein (biological macromolecule) is the basis of my University Scholar project.
Summer 2006
This summer I will obtain funding to remain at UConn and carry out research under the supervision of Dr. Birge. I will also take the two gen. eds. I am not able to fit into my regular schedule because of the graduate courses I plan to take. This includes the group 4 and a group 5a requirements based on what courses are available during the summer sessions.
Fall 2006
Standard Plan of Study University Scholar Plan of Study
Chem 234 Instrumental Analysis (4) Chem 234 Instrumental Analysis (4)
Chem 296 Undergraduate Research (3) Chem 296 Undergraduate Research (3)
Elective (Group 6) PLSC 246 Biotechnology (3)
Elective (Group 7) Chem 360 Biological Chemistry I (3)
Elective (Group 4 or 5a) MCB 338 Techniques in Structural Biology (2)
ARE 110 Population, Food, and the Environment (3)
For requirements, I will take Chem 234, PLSC 246, and ARE 110. I also will take undergraduate research and two graduate courses. I plan to take Biological Chemistry I (Chem 360) and Techniques in Structural Biology (MCB 338). Both of these courses will provide me with a good background in different areas of biochemistry one focusing on biology techniques and the other focusing on the mechanisms of chemical processes in biological systems. Both of these courses will enhance my University Scholar project and will help me upon graduation.
Standard Plan of Study University Scholar Plan of Study
Chem 214 Intermediate Inorganic (3) Chem 214 Intermediate Inorganic (3)
Chem 215 Inorganic Lab (3) Chem 215 Inorganic Lab (3)
Chem 297W Senior Thesis (3) Chem 297W Senior Thesis (3)
Chem 296 Undergraduate Research (3) Chem 296 Undergraduate Research (3)
Elective (Group 4 or 5a) Chem 393 Spec. Topics in Physical Chem. (3)
Chem 361 Biological Chemistry II (3)
For my requirements I will take Chem 214, Chem 215, Chem 297W as well as undergraduate research. The two graduate courses I plan to take include a course in physical chemistry taught by Dr. Birge (Chem 393), which will either cover molecular modeling or electronics depending on which is offered, and Biological Chemistry II (Chem 361). Chem 361 will enhance what was learned in 360, and the special topics course will provide background in an area I have no experience but is often used in our laboratory.
If any of these graduate courses are not offered or conflict with other required courses, other courses I would take include; Chem 343: Organic Reactions, MCB 312: Foundations of Struct. Biochem., and MCB 308: Theory of Biophysical Techniques.