BioacTiVity: Dog-tor Lilo’s in Charge

Welcome back! If you don’t remember me from last summer, my name’s Lilo and I live on campus with my mom, Micaylah. If my name doesn’t ring a bell, you might recall seeing me this past semester where I spent a shocking amount of time in my rain boots and taking casual dips in the fountain.

Since it’s my second summer now, I guess you could say I’m the top dog around the lab. My days are pretty busy, especially since we had three new humans join us this year. Thankfully, that’s given me lots of practice explaining the research we do here, so allow me to give you the rundown.

The Buettner lab does bioinorganic chemistry research, which means they study metals in biology. More specifically, the lab looks at interactions between metals and proteins. Now don’t get your hopes up, they don’t study delicious proteins like chicken and beef, they study Due Ferri (DF) proteins, which were computationally designed to bind two irons. With five of us in the lab, it gets a little busy, so follow me for a crash course on the projects we have going this summer.

With five of us in the lab, it gets a little busy, so follow me for a crash course on the projects we have going this summer.

Project 1: The Power of the Dog Binding

Since we can’t dive into that masterpiece of a movie now, let’s see what Ethan is working on! Some days when he is too lazy to run, he’ll join me and Micaylah on our morning walks, so through dogspeak, he told me that he loves to do puzzles with his friends and enjoys watching the latest Marvel shows/movies. Thanks to the X-SIG brown bag lunches, he’s getting better at explaining his research, so let me walk you through what he does.

Ethan focuses on the six DF protein variants of the Due Ferri single chain proteins. He is following up on the work Brittany was doing last summer, and is focused on a set of six DF protein variants, three of which (DY, DFY and G4Y) have tyrosine, a hard Lewis base, in their active sites. He is looking to see if the substitution of this tyrosine improves the binding interactions of the DF variants and his metals of interest, titanium, vanadium, and zinc. His main goal is to characterize metal binding of their mini-metalloenzymes. His research not only helps them better understand the role metals play in biology, but also aids in the design and development of new enzymes!

Oftentimes, you will find him by the circular dichroism spectrometer (but you’ll only hear it as CD) where he can monitor the secondary structure of the protein.The CD helps Ethan understand the secondary structure of the protein. Since the de novo proteins he uses have 4 alpha-helices, he can watch the peak at 208 and 222 nanometers as he increases the concentration of zinc. We can see the intensity increases at those wavelengths as the amount of metal is increased. until all the binding sites become fully saturated.

Here is a CD titration graph of one of our proteins, G4Y, as he continues to increase the concentration of zinc in our samples. The first line is the protein without any metal added, so you can see that as he increases the equivalence of zinc, the alpha helical structure increases. 

When he looks at just 222 nm to get a better understanding of the saturation from each addition. Even my dog eyes can see that nice curve down as more zinc is added! This indicates a flattening out of metal binding as the protein’s active site becomes saturated with zinc.

We’re halfway through and it seems like he’s got his assay conditions down, so now we focus on replicating! Right now, Ethan started working with zinc and recently started experimenting with vanadium! He looks forward to future experiments, especially with vanadium (and hopefully some titanium). I’m sure he’ll tell me all about it on our morning walks, but I’m good at tuning him out. Speaking of, that’s enough of him!

Project 2: Small but Mighty

This is my mom, Micaylah. She’s a BMB major and she’ll be starting her senior year this fall. In her free time, she likes taking me on long walkies, playing Yahtzee (a LOT of Yahtzee), and binge watching Harry Potter.

Last semester, she went to the American Chemical Society conference in San Diego (and left ME behind…how rude!) with her past lab mates, Brittany and Oliver, where they each presented their research from the past year in a poster session. Brit and Oliver just graduated, but before they left, they made sure me and my mom knew it was our job to train the new students to be just like them. We’ve done a fine job, if I do say so myself!

Her research focuses on testing how well their metalloenzymes work. Specifically, she uses two different small molecule assays to assess the kinetics of phosphate bond cleavage. This is to follow up on some work that was done in the past, where the lab has seen that these proteins cleave DNA, but they’d like to get a more quantitative understanding of what is going on. This past semester, she started working with a small molecule called DiFMUP. When her metal bound enzyme cleaves the phosphate bond in DiFMUP, it fluoresces.

The hope is that when she adds titanium (or another metal) to her protein, the reaction happens faster and more of the fluorescent product is produced. The figure on the right is some of data my mom collected last week from her DiFMUP assays. The first few data points of each line, or the initial rates, are the most important. She compares these initial rates between samples to quantitatively determine how good of an enzyme we have.

This summer, she’s also working with another small molecule, Bis(4-nitrophenyl) phosphate, or BNPP. This reaction is essentially the same, but when the phosphate bond of BNPP is cleaved, the product is yellow. Last summer, she spent lots of time monitoring this reaction with UV-vis spectroscopy, but this summer, she’s monitoring it using phosphorus NMR spectroscopy. She hopes to see that as the reaction proceeds, the peak for the reactant (BNPP) should slowly disappear, and the peak for the product (PNP) should start to form. Ideally, the metal bound proteins should show product formation at a faster rate than protein alone.

By the end of this summer, my mom wants to fit her data to the Michaelis Menten equation, which is the holy grail of enzyme kinetics. If we can figure this out, the paw-sibilities are endless! Things are running pretty smoothly this summer with me as the branch manager (they don’t call me that just because I like to chew on a good stick), so I definitely see that happening in her near future.

Project 3: Attack of the Fluorescent Proteins!

Hey look, it’s Cristin, or CMac as Ethan calls her! She is a rising junior majoring in BMB with a minor in neuroscience. She loves having me around because I remind her of her dogs (except I’m cuter…. obviously). She spends a lot of time either crocheting or walking through the battlefields. She was really nervous for the lab this summer because she is not familiar with inorganic chemistry but since Dr. Buettner and my mom have been such good teachers, she is becoming much more comfortable in the lab.

Cristin is always on the move (she might walk even more than me!). She is the protein prepper in the lab, which is a pretty big job considering there’s three other people whose daily experiments depend on the proteins she makes! She preps all of the proteins in the lab. She grows them with E.coli, purifies, and freeze-dries them to make sure there is plenty to go around!

Micaylah and Brittany spent time two summers ago finding common amino acid residues in the active sites of naturally occurring vanadium enzymes. They found that most vanadium enzymes contained arginine residues in the active site. From there Oliver redesigned the G14D protein (that Micaylah and Ethan work on) to substitute a glutamate residue with an arginine residue. There are three glutamate residues in the active site that he chose to focus on, positions 11, 44 and 74. The Buettner lab’s arginine protein series contains single, double and triple arginine substitutions that make a more positively charged active site.

Cristin is currently using the arginine proteins to find a correlation between vanadium concentration and inherent protein fluorescence. I hadn’t heard of a fluorimeter before so she explained it to me. Some amino acids fluoresce so when they are present in proteins the fluorescence can be measured.

When metals bind to the proteins, changes in the structure of the protein (like Ethan talked about!) and changes to the pull on the electrons can change the fluorescence, so Cristin is trying to see if she can monitor metal binding using fluorescence in the arginine proteins. She is working with zinc as well as two different types of vanadium (ammonium metavanadate and vanadyl sulfate). All of her samples contain the buffer, proteins and different concentrations of metal. She runs all of her samples through the fluorometer and then scales the data based on the peak found in the protein alone sample to see how the peaks vary upon the addition of metal.

She also uses the CD like Ethan, but she uses it to do protein melts. These melts are used to determine the thermal stability of a protein under increasing temperature conditions. Since the arginine proteins are designed to bind well to vanadium, we would expect to see an increase in thermal stability as vanadium is added. Heating a protein up (like chicken, my favorite) causes it to denature. With her protein melts, she would expect that these vanadium bound proteins would stay in their coiled state longer than the proteins alone. Unfortunately, these melts do take up the whole lab day so she has only been able to do one so far. 

Cristin’s goal by the end of the summer is to be able to further biophysically characterize all the DF proteins she preps. She really likes research in the Buettner lab and hopes to see some paw-sitive results soon.

Project 4: Remain paws-itiVe

Now it’s Audrey’s turn! Audrey will be a sophomore this fall, and she is majoring in Chemistry. She is on the track and field team here at Gettysburg, and much like me, she loves to spend her days going on walks, runs and being outdoors. She also likes to play games with the lab, although she said it gets pretty intense when they all play together! Audrey has learned a lot this summer since it is her first time doing research.

In the lab, Audrey has been working with the arginine proteins Cristin talked about to create functional vanadium enzymes. In nature, these enzymes often function as something called a  haloperoxidase. Although vanadium is a hydrolysis-prone metal, these haloperoxidases are able to carry out their reactivity in water. By utilizing the DFsc protein system, Audrey has been trying to mimic these enzymes. She has been doing this by creating assays which allow her to monitor the activity of the vanadium enzymes. Audrey has been using a few different instruments to help further her research this summer.

Some days you will see her sitting in the NMR room running samples on the UV-visible absorbance spectrometer. This instrument measures the light that is absorbed by the samples at each wavelength. This is useful because it helps Audrey measure the enzyme’s kinetics and gives her a better understanding of what is happening with the peroxidases.

The enzymes that Audrey is measuring with this instrument include arginine proteins, hydrogen peroxide, vanadium, buffer, and a substrate called thionin. The chemical reaction of these enzymes is initialized with the addition of the thionin. This reaction then proceeds to bleach, so it begins as purple when the thionin is added, and then loses its color over time.The graph on the right was made with data collected from the UV-vis. Because this reaction is slow, the samples used to create this graph were run on the UV-vis the next morning after they were made. As you can see, the protein-metal complexes have a high absorbance all at the same wavelength, 600 nm. This is because thionin has an absorption at 600 nm. The data shown in this graph is what we would expect to see because this is a bleaching reaction. So, the sample with just the substrate has the highest peak, as it does not lose its color, and the other complexes have a lower peak, because they lose their color over time. When the thionin is added to the protein and the metal, it begins to bleach since it is being catalyzed.

Audrey also has been reading a lot about electrochemistry recently, as she is beginning to work with cyclic voltammetry (CV). While running the CV, Audrey is looking to see what the redox potentials of the vanadium enzymes are. As the summer continues, Audrey hopes to make more progress with her enzymes, and conduct more CV. Sometimes research can be difficult for Audrey because she doesn’t always get the results she wants, so it is my job (as the leading dog of the lab) to remind her to stay paws-itive and be optimistic!

Well that was fun, but I’m exhausted after that long day in the lab so it’s time for me to take a nap next to my window. Anyways, I hope you enjoyed our walk through the Buettner lab! Come by soon for snacks and silly pictures of me, my mom takes several thousand a day!

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