MSTF Researchers: Judy Cichoracki and Jared Seaman

Overview of current research:

Natural metalloproteins can perform elaborate functions with high selectivity under mild conditions, yet the range of processes they carry out is limited to biologically relevant functions. A deep understanding of the relationship between protein structure and function will permit the preparation of artificial proteins tailored to specific non-biological applications. This ability would have a significant impact in the design of biosensors, enzymes for non-biological transformations, and smart materials with high levels of spatial control in the meso- to nano- scale. In this context, membranes and membrane-associated proteins are particularly relevant, as the lipid bilayer provides a pre-organized 2D structure.
We have engineered a heme-binding site into a stable, membrane soluble scaffold. This initial design, ME1, binds ferric protoporphyrin IX with high affinity, exhibits a redox potential within the range of natural membrane heme-binding proteins, and shows nascent catalytic activity
ME1 represents a suitable model system for the study of structure-function relationships in membrane protein
We are currently exploring the importance of the heme environment, its hydrophobicity and the presence of aromatic residues in determining the midpoint potential and the binding affinity within the context of our model membrane protein. This investigation will also address the effect of mutations on the binding affinity for both the oxidized and the reduced state of the iron porphyrin.

In parallel, we are optimizing a nascent catalytic activity observed in our first generation design. We designed a 5 coordinate mono-His ligated analog of ME1, in which the second axial position is available for oxygen activation and/or substrate binding.

After a HPLC break down we are focusing on adding a cystiene to the end of the mutant chain R-G25F-GG-Fmoc to get a new mutant chain R-G25F-GGC. Our first step was to remove the Fmoc (9//H//-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl) from the origional chain using a basic solution of 20% piperididne in Dimethyl formamide (DMF).

Example of Fmoc cleavage
This was reacted for 2 x 1 hour reaction baths to remove the Fmoc. After this step we had a chain that contained an amine, R-G25F-GG-NH2. We removed the amine group and replaced it with a cysteine by reacting the chain with a cysteine mixture with activators (152.8 mg HBTU in 802 microliters DMF, 660 microliters NMP in 330 microliters DIEA, 236.5 mg cystine in 2 mililiters DMF). This was reacted for 4 hours.
We tested the reaction for the presence of amines to get a qualitative confirmation that the amines were not present. This was done with the Keiser Test. We added a cocktail of specific chemicals and let react at 70 degrees F for 5 min. If it turns blue/indigo then amines are present. Our sample was free of amines. We are now doing a mini-cleavage to remove the Fmoc from our sample and test it for the GGC or GG sequence.

Once we completed the mini-cleavage, we looked at the molecular weight to determine if we had what we thought. The molecular weight was right where expected.
We were unable to run the sample through the HPLC to purify it. The pump went down and is awaiting repair.

The current hemochrome test uses heme dissolved in a phosphate buffer (pH 7). The heme is oxidized with a standardization solution (.2M NaOH, 20% pyridine, and 2μL/mL K3Fe(CN)6 . Absorption at 540nm is detected. The heme is then reduced with sodium dithionate and absorption at 557nm is detected. The heme concentration in the cuvette is calculated using the following formula:
( abs. @557nm - abs. @ 540nm) =
24.04 mM

The new hemochrome test uses heme dissolved in the same phosphate buffer. An acid solution (composed of 66.5% ethanol, 17% acetic acid and 16.5% water) was added to the heme solution at a 1:1 ratio and the absorption at 398 nm was determined. The concentration in the cuvette was calculated using the following formula:
Beers Law: Absorption = εbC
Where ε is 144,000, b is 1 cm, C is the concentration in mM.

ME1 peptide titration using the old heme-chrome test. .02 μM heme in 1.2 mM DPC (detergent). Absorption at 414nm was detected as the concentration of peptide was increased by 1 μL of 100μM peptide solution.

ME1 heme graph is in link above

Classroom connections

Students will construct berry powered fuel cells. After construction they will take them into the sun to charge them. They will then store the energy in a capacitor to later compare with the time it took to charge the cell with the time it takes to use the energy in the capacitor with a small motor. They will measure the voltage and amps with multi-meters and compare that to the battery power. They will then talk about what this technology could be used for on a larger scale
Students will test solutions containing Fe+2 and Cu+2 ions for absorption of the UV-Visible spectra. They will compare the results with data obtained from mole conversions and calculating molarity. Students will also measure emission spectra from metal salts that are energized with burning methanol. The spectra obtained will be related to the electron configuration for each of the elements tested.