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> CBIN Home> Education > University > Research Projects
2007 CBIN SURF Research Projects
Student, Undergraduate Institution
MSU Faculty Mentor, Department
Geoff Blatter, Montana State University
Brian Bothner, Chemistry & Biochemistry
Emily Buckhouse, Montana State University
Mark Young, Plant Sciences & Plant Pathology
This summer I have been working with the g41c Heat Shock Protein. Essentially what I am trying to do is create a protein whose structure remains relatively stable even after I have labeled select lysines on the surface of the cage. The reason we are interested in lysines is because we can attach targeting agents to the surface of the cage through the lysines and we can use cysteines to attach drugs to the interior of the protein cage. One of my main goals is to keep the interior of the protein cage separate from the exterior of the cage. I want to ensure that our targeting agents stay on the exterior and the drugs stay on the interior. To begin looking at lysines that we could attach targeting agents to, I had to determine which lysines will label with an NHS-Ester. I used mass spec to analyze this data and found that 4 lysines label. Now what I have to do to ensure that the stability of the protein remains present is mutate all of the lysines that contribute most to the stability of the protein, such as those lysines that are involved in salt bridges. The lysines that are involved in salt bridges will be changed to arginines, which are similar in structure but different in reactivity, therefore they will not label. Controlling which lysines we label will also allow us to know exactly where the targeting agents are on the exterior of the protein cage. I will start mutating this protein in the next couple of weeks.
Krysta Buska, Montana State University
Trevor Douglas, Chemistry and Biochemistry
Catalase Activity in SsDps
Optimal pH Determination
pH controls
Testing activity at various concentrations
Peroxide loading (8 to 1024 H2O2/subunit)
Protein concentration
Temperature (room and 65oC)
H2O2 quantification using KMnO4
Lineweaver Burk Plots
Cameron Chen, Montana State University
Yves Idzerda, Physics
The Idzerda Lab has recently aquired a SONOPLOT microplotter capable of depositing a femptoliter sized volume in a highly repeatable fashion. Fluid that is to be deposited is held in a micropipette with a 10 nm aperature (different sizes are available). To expell fluid onto a surface, the aperature is brought to within 1 micron of the surface and is vibrated by means of a peizoelectric crystal at a frequency of 450 Khz. This frequency represents a fundamental frequency of the micropipette, whose walls flex in and out at the antinodes created in the glass wall of the micropipette. This flexing produces a pumping action, resulting in deposited fluid onto a substrate. The amplitude and duration of the frequency pulse are completely programmable, allowing precise volumetric control of the deposition. In the near term it is anticipated that this microplotter will be used to pattern biological materials developed by CBIN. However, microplotting is an art and many parameters need to be evaluated before repeatable function is possible.
My responsibility this summer was to drive meaningful progress with regard to useability of the device. To demonstrate the utility of the microplotter, we are fabricating micron sized lenses snd demonstrating their functionality by using these dust sized lenses to efficiently couple light from one fiberoptic cable to another. In addition to possible commercial applications, we can use this time to improve the microplotter with regard to making upgrades to software, hardware and our protocol as appropriate.
The protocol and materials used to fabricate microlenses are relatively simple. A liquid, UV curable resin is deposited onto a glass surface. Wetting properties on resin on glass dictate a spherical shape of the droplet. UV light hardens the resin and creates a plastic lens. Resin is easier to plot that aqueous solutions because it evaporates only very slowly. The simplicity of this protocol allows us to refine the microplotter as a device that will one day be able to handle biological materials.
Joe Fox, University of Montana
John Peters, Chemistry & Biochemistry
During the past 12 weeks I have been working with Drs. Anatoli Naumov and Oleg Zadvorny in John Peters’ lab on two different projects. The first, with Dr. Naumov, was to clone and sequence the purple sulfur bacteria Lamprobacter Modestohalophilus to compare the genetics of its stable hydrogenase enzyme to those previously discovered. Currently, the cloning is complete with the sequencing still in progress.
The second project, with Dr. Zadvorny, was to use nickel nanoparticles as a catalyst in a model hydrogenation reaction. In a toxic nickel environment, the hydrogenase enzyme can detoxify the nickel, forming Ni(0) nanoparticles 5 nm in diameter. Overall, the goal is to use the hydrogenase enzyme to evolve hydrogen gas, given proton and electron sources, which would then be used to hydrogenate a substrate, using the nickel nanoparticles as a catalyst. Thus far, I have used the procedure of Xu et al. (Nanotechnology 18 2007) to synthesize nickel nanoparticles to use as a positive control. Transmission Electron Microscopy data has confirmed that these particles are indeed nickel, but are of varying sizes. Most recently, these nanoparticles have shown nearly 100% hydrogenation of acetylene to ethylene. I would like to thank both Dr. Naumov and Dr. Zadvorny for their help in these projects, the Center for Bio-Inspired Nanomaterials and Dr. John Peters for the opportunity to work in an outstanding lab.
Logan Giles, Earlham College
Robert Szilagyi, Chemistry & Biochemistry
Electronic structure studies of iron-sulfur clusters from FeFe-hydrogenase
The FeFe-hydrogenase contains four Fe4S4 clusters (terminal, distal, proximal, and part of the catalytically active H-cluster) with cysteine residues connecting them to the protein. Upon initial calculations of the “distal” cluster, three distinct energy levels were obtained depending on which two iron-sulfur rhombs were coupled antiferromagnetically. Further investigations were taken into the origins of these energy levels by first fully optimizing the cluster and then probing various constrained bond distances and angles. The energy levels were found to be little influenced by thiolate ligand-iron distances as moving the thiolate ligands farther away or closer did not separate the energy levels considerably. However, the iron-sulfur distances showed the most influence on the separation of the energy levels. Shorter iron-iron bonds within the ferrocoupled rhomb corresponded to a lower energy structure, which implies that the iron–sulfide sulfur distances have to be elongated. We also find that the iron-sulfide sulfur bond distances within antiferrocoupled rhombs has to be short in order for the sulfur to be able to sufficiently donate electron density to the iron. The dependence of the energy levels and the magnetic interactions on bond angles are currently underway.
Kevin Harlen, Montana State University
Trevor Douglas, Chemistry & Biochemistry
Optimizing conditions for SIRV (Sulfolobus islandicus rod-shaped virus) purification.
This summer I worked on purifying large amounts of protein and concentrating it to run on a 96 well plate. The plate will have different amounts of protein concentrations as well as varying pHs. We are looking for the concentration and pH that will cause the protein to self assemble into fibers. We will then characterize these fibers and attempt to mineralize them using different metals.
Kassy Lynass, Montana State University
Mark Young, Plant Sciences & Plant Pathology
Testing a method using a cation exchange column with hexanediol wash 6to separate endotoxin from heat shock protein.
Tim Potter, Montana State University
Brian Bothner, Chemistry & Biochemistry
The goal of experiments I conducted was to observe digestion of the DPS protein under different reaction conditions. However, minimal digestion was seen through the MALDI and little to no digestion was observed through SDS-PAGE gels. We began to use the protein's sequence to map what peptide fragments were observed from each digestion. Using MALDI and SDS-PAGE gels, I ran many DPS digests adjusting different reaction conditions like temperature, addition and different amounts of Urea, and different timepoints to try and find optimal conditions for digestion. So far most of the results have been the same, although I plan to run several more digestions with increasing temperatures to see if the protein becomes less stable at a higher temperature. I also ran several experiments with the fluorometer and UV spectrophotometer to increase my knowledge of the instruments available on campus.
Courtney Reichhardt, Montana State University
Trevor Douglas, Chemistry & Biochemistry
Project Goals:
- Use mineralized Bacterio ferritin protein to catalyze hydrogen gas production;
- Optimize conditions of mineralization of Bacterio ferritin with platinum
- Incorporate additional porphyrin into the shell of Bacterio ferritin protein
Future Project Goals:
- Try to incorporate other transition metal porphyrin into Bacterio ferritin protein and
- Mineralize Bacterio ferritin that has had additional porphyrin successfully incorporated into its protein shell
Ellie Rudy, Montana State University
Mark Young, Plant Sciences & Plant Pathology
Kelley Thornsberry, Montana State University
Trevor Douglas, Chemistry & Biochemistry
This summer, my project was to create a coordination polymer (a complex
of metal ions and ligands) inside a protein cage. I worked mostly with heat shock protein that had 3 amino acids per subunit substituted with cysteine. Since cysteine has an SH group, it can be bonded to a ligand inside the protein cage, and it is from there that the coordination polymer will start to assemble. The main idea is to coordinate an MRI contrast agent, a fluorescence agent, or pharmaceutical agent inside the cage and attach some kind of targeting molecule to the outside of the cage, so that the contrast agent, drug, etc. will go where it needs to be. Less of the agent will be required since the protein cage has targeting molecules, so one advantage would be decreased side effects. The metal I worked most with in creating a coordination polymer was iron II, and that proceeded well. I recently began working with the metal, gadolinium, which is useful as an MRI contrast agent.
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