Molecular Biology

1. Molecular Biology By: Kianna Ferguson University of Florida At the Institute for Molecular Biophysics (IMB) at Florida State University

2. Project Objective • The Troponin complex regulates muscle contraction by preventing myosin and actin from binding and thus contracting. • Our eventual goal is to see if the absence of Ca2+ will change the conformation of the protein Troponin I • We’re hoping for a change but a result of • no change is still a result 

3. Troponin • Troponin is a regulatory protein complex • It actually controls the position of the tropomyosin which blocks the actin/myosin interaction • The troponin complex is made up of three proteins: • Troponin I (TnI), Troponin C (TnC), Troponin T (TnT) • The troponin we use in lab is a mixture of the cardiac troponin from mice and from rats • e.g. Troponin I – rat ;Troponin C - mouse

4. Troponin Complex • The blue-teal helices are TnI • The red helices are TnC • The yellow helices are TnT

5. Troponin without and with Ca2+ - Ca +Ca TnI is yellow TnC is red TnT is green The switch peptide makes contact with the TnT and TnI coiled-coil

6. Steps of Overall Experimentation • There are 5 major steps to our experiment • Step 1: Mutate the DNA of the protein • Step 2: Grow and express the protein • Step 3: Label the protein • Step 4: Use the EPR technique • Step 5: Analyze the Results • I mostly work on the first 3 steps

7. My section of the Experiment • Step 1: Mutate the amino acids of the protein • Remove all the cysteines in the Troponin I protein • Add two cysteines in specific places • 129th amino acid and 160th amino acid • We do this by designing primers with the desired mutations and extending the DNA strand to have those mutations

8. Step 2: Growing and Expressing Troponin • This is my area of concentration • There are six main processes in this step • First we do a small scale grow up • We take small amounts of a frozen stock of our expressed Troponin I and place into a flask of 5 mL of media • Then we incubate and grow bacteria with the proteins included inside the cells

9. Step 2: Growing and Expressing Proteins- part 2 • Next is the large scale grow up • In this step we increase the volume of media in which the E-coli flourish to 1 Liter • The third step is lysing the cells • We use the microfluidizer

10. Pictures

11. Step 2: Growing and Expressing Proteins- part 3 • The fourth step is dialysis • This means we saturate the protein with a buffer to achieve a specific pH. • TnI: pI is 9.50 and desired pH is 8.0 • The fifth step is running a column • In this step we purify and separate our desired protein from everything else. • We collect all the purified protein in tubes to later be concentrated and labeled

12. Column Pictures • This is the column •  These are the collection tubes

13. Graph from a Column Using the spectrophotometer, we can determine the concentration of the protein and in which collection tube it is The pink line is the conductivity of the sample. The blue is the absorption, which is the concentration of protein.

14. Step 2: Growing and Expressing Proteins- part 4 • After the column is complete, we run a gel This is TnC-15; made as part of the Troponin Complex

15. Steps 3: Label the Protein • This step is preparation for the EPR (Electronic Paramagnetic Resonance) technique. • Here we label the protein with spin labels: MSL or MTSSL • This is in order to secure a signal by the EPR technique.

16. Step 4: Electronic Paramagnetic Resonance Technique • This is the stage of the experiment to determine the extent of the change, if any of the conformation of the Troponin I • The distances between the two cysteines as calculated by this machine will tell how much, if at all, the protein has changed.

17. EPR Machine Pictures

18. Lab People!!! • Mr. Jean Chamoun • My direct boss • Person I work closest with • Dr. Peter Fajer • My mentor and family friend! • Mr. Clement Rouviere • Lab Boss! • He’s gone and we are SAD!! • My whole lab! • Very diverse and fun!

19. Acknowldgements- II • The ever so helpful REU staff • Dr. Pat Dixon- for making sure I got paid by somebody • Ms. Gina LaFrazza- for tolerating my incessant e-mails and for paying me • Ms. Stacy Vanderlaan- for all the trips to Wal-Mart • Mr. Carlos Villa- for loaning me a disk once

20. Any Questions?

21. Step 4: EPR continued • The EPR technique detects the distances between the labeled cysteins • There are three catergories of EPR that we use • Electronic Coupling: under 8 Angstroms • Dipolar Coupling: 10-20 Angstroms • DEER: 20- 50 Angstroms • If it is larger than 50, we are to assume that no appreciable change has taken place as our original distance was greater than 50 A

22. Step 5: Analyze the Data • In this step, we simply compare any movement of our double mutations to our ‘control’ • Our control is a single mutation (at 160) • This makes sure that the changes we get are only between the cysteines in each protein as opposed to in between cysteines of different proteins

24. Scanned Gel • This is a gel of TnC-15. • I made this as part of the Troponin Complex.

25. Troponin’s 2 states • Jean’s with and sans calcium pics