Protein Structure

Yesterday (January 29th), I went to RPI for more interesting analysis! We started with a summary of protein structure, which was really cool because we recently learned about protein synthesis and structure in AP Bio, so it was really cool to apply it to research! Protein structure is divided into different levels of structure. Primary structure (1˚) consists of the amino acid sequence of the protein. To form secondary structure (2˚), different amino acids that are not adjacent in the sequence can react and cause twists in the amino acid strand. The two common secondary structure forms are the α helix and the β pleated sheet. Today, as I will explain later, I focused on α helices. Tertiary structure (3˚) is formed when the secondary structure folds on itself, forming a three-dimensional structure. Finally, the quaternary structure (4˚) gives the protein form and function by adding molecules such as sugars and phosphates or combining different proteins together. Below is a diagram showing the different protein structures.

Source: Madison Technical College Lab Manual on Protein Structure

Today, my task was to look at a protein structure using MOE software. We are looking for a site to bind another molecule, especially the α helix sites. Two α helix forms can aggregate like a zipper, like the bindings of two spiral notebooks. I identified all of the different helix sites and recorded them by amino acid sequence numbers. After they were identified, I color coded them on the model so we could see where they were located in the three-dimensional surface structure of the protein. It was so interesting to see the relationship between the amino acid sequence and the final structure of the protein! It added a lot to my knowledge of protein structure and allowed me to apply something I learned in class.

As usual, I can’t wait to see what comes next!

Analyzing Data!

Today (January 22nd), I went to RPI for another day of interesting work! Today, I analyzed the data from the experiment I was introduced to on December 11th. To summarize the experiment, we were analyzing the size of particles that can normally pass throught the blood brain barrier, using a set up with two wells, one inside of the other (seen below). Twenty wells were tested. Ten had the dextrans added to the apical (inner) chamber, two each for the five different dextran solutions (4 kDa, 10 kDa, 20 kDa, 40 kDa, and 70 kDa). The other ten had the dextrans added to the basolateral (lower) chamber, two each for the five different dextran solutions. Further explanation of this experiment is given in my blog post from December 11th.



The data from this experiment came from solution samples taken at various times over a 25 hour time period. These samples were then scanned and analyzed according to their fluorescent signal. The dextran molecules contained Fluorescein Isothiocyanate (FITC), so the more fluorescnet signal the sample had, the greater the concentration of dextrans in that sample.

To analyze the data, I copied the data from the scan program into a program called Origin. There were three separate scans, so I had to average the data from the three. I then plotted the data for Concentration vs. kDa. After detecting errors in the beginning and end of the graphs, I trimmed the graphs to get a more accurate linear fit. After trimming the graphs, I calculated the r-squared values for each kDa. 4 kDa had by far the best fit. 10 kDa had the worst, and then the r-squared values increase for the 20, 40, and 70 kDa. After examining the Concentration vs. kDa data, I plotted the data for Concentration vs. Time for each of the 20 wells. For every well, the concentration increased over time. This is because as the time increases, more of the cell monolayer dies off, and more dextran molecules are allowed through the barrier. From the starting concetration values, it was evident that the larger molecules started with a lower permeability.

Overall, I had a very interesting day of analyzing data! I can't wait to see what's coming next week. 

Passaging and Protein Structure

Today (January 15th), I went to RPI for my first day of lab work of the new semester! Today was full of really interesting new things. First, JP showed me how to culture tissue, particularly the practice of passaging. Passaging is the process of moving some cells from a previous culture to a new growth medium to allow them to continue to grow. The first thing we had to do was spray everything down with ethanol. Everything must be sterile when working with the tissue, so all materials were sprayed, including our gloves. Next, we got the tissue culture out of the incubator, which maintains the CO₂ level and temperature similar to that of the human body. Before passaging the tissue, I was able to observe the culture under the microscope. The proteins that had been previously cultured were connected in a chicken pattern, and they were attached to the surface of the container with linkages that kept them in place. First, the media was removed from the container with a vacuum line. Then, 10 mL of PBS solution was added to the container to remove dead cells and remove the Fetal Bovine Serum (FBS) that inhibits the enzyme Trypsin from acting on the cells. Trypsin functions to cut the anchor linkages that connect the protein to the container, causing them to float around, which is not wanted while the tissue is being cultured. The PBS was then removed from the container with the vacuum line. Next, 1 mL of Trypsin was added to detach the cells from the container. After the Trypsin was active in the container for a few minutes, I observed the cells in the container again, and they were now floating islands of groups of cells. Next, FBS was added back into the container to re-inhibit the Trypsin. This mixture was then transferred to a conical vile to be spun down in the centrifuge. Once the centrifuge was balanced, it was spun at 1000 rpm for 5 minutes to make a protein pellet separate from the media. While the vile was being spun, we prepared a new container, and labeled it as passage #25 of the cells. 35 mL of new media were then added to the new container. After the vile was done in the centrifuge, the old media was drained from the vile using the vacuum line. We then added 1 mL of media to the vile to re-suspend the protein pellet by using the pipette up and down until the protein pellet broke up and dispersed in the media. 100 microLiters of the suspension were then added to the new container, and it was agitated to spread the cells around the surface of the container. The passage was then complete, and we re-cleaned everything with ethanol and put the new culture in the incubator.

After passaging, JP introduced me to a new computer program for modeling proteins. The program displayed both the amino acid sequence and the three-dimensional structure of the protein. My job was to search through the entire amino acid sequence to highlight certain amino acids that are important to the function of the enzyme. Once I highlighted the amino acids in the sequence, I also highlighted the corresponding structures on the enzyme that those amino acids code for. The purpose of this activity was to show the position of the binding site of the protein structure. After locating the binding site and seeing its structure, we looked at how the signal molecule (which is a sugar) binds to the binding site. This program was really interesting because I am learning about protein synthesis and structure in AP Biology right now!!!!

I can't wait to return next week and find out what's next!

Cleaning Up

12.18.12

On Tuesday December 18th, I went over to RPI for the last time before winter break. Because RPI was wrapping up their work as well, there were not any new experiments to be set up or new data to interpret. My day was full of cleaning and organizing the lab equipment and bench spaces. Also, I was able to see our lab's liquid nitrogen container, which was really cool! I can't wait to return after break and continue with our work.