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141214-490 Presentation
- 1. Characterizing the heme pocket of Tt H-NOX using unnatural amino acids Lukasz T. Olenginski and Christine M. Phillips-Piro Franklin and Marshall College Department of Chemistry
- 2. A Sensitive and Tunable Gas Sensor Heme Nitric Oxide and Oxygen binding (H-NOX) proteins Thermoanaerobacter tencongensis (Tt H-NOX) Hydrogen-bonding network Y140 Critical residue: PDB ID: 1U55 Heme containing Fe2+ binds: CO, O2, and NO
- 3. Tune O2 Binding Affinity Incorporate UAAs at Y140 in order to tune O2 binding affinity O O His H O Fe O O His H O Fe N O His H O Fe Stronger H-Bond donor Weaker H-Bond donor H L-3-Nitrotyrosine L-4-Aminophenylalanine (mNO2Y) (pNH2F) Tyrosine NO O
- 4. In vivo Incorporation of UAAs Gene of Interest tRNA + Aminoacyl tRNA synthetase Minnihan EC, Yokoyama K, Stubbe J - F1000 Biol Rep (2009) tRNA synthetase RIbosome tRNA tRNA with UAA Protein with UAA
- 5. SDS-PAGE gel of Tt H-NOX with UAAs L-4-Aminophenylalanine (pNH2F) L-3-Nitrotyrosine (mNO2Y) N H H HO N O O
- 6. SDS-PAGE gel of Tt H-NOX with UAAs L-4-Cyanophenylalanine L-4-Chlorophenylalanine L-4-Bromophenylalanine L-4-Iodophenylalanine pCNF pClF pBrF pIF Cl Br IC N
- 7. Further Mutate the Heme Pocket PDB ID: 1U55 Y140 F78 OH O NH2 OH O NH2 SDM F78A Y140 A78 Phenylalanine Alanine
- 8. Goals for Next Semester Further mutate the heme pocket of Tt H-NOX Incorporate UAAs at other sites in Tt H-NOX Assess enzyme function upon UAA incorporation in T4 Lysozyme
- 9. Probe Other Sites in Tt H-NOX Incorporate UAAs at different sites Assess protein structure, stability, and function with different UAAs PDB ID: 1U55
- 10. Assess Enzyme Function T4 Lysozyme PDB ID: 148L Assess structural stability/enzyme function upon UAA incorporation T4 Lysozyme Y24 Y18 F67 F153
- 11. 180 ° Assess Enzyme Function PDB ID: 148L Residues near substrate binding pocket are solvent accessible Y24 Y18
- 12. Acknowledgements Dr. Scott H. Brewer Gregory Olenginski, Elise Tookmanian, Nicole Maurici Lisa Mertzman, Julie Gemmell Dr. Christine M. Phillips-Piro Hackman Scholars Program Marshall Scholars Program People: Grants: Research Corporation Grant Franklin & Marshall College Department of Chemistry NSF (CHE-1053946) to S.H.B.
Editor's Notes
- Trying to see how UAAS affect gas affinity (via UAA at a variety of sites) and enzyme activity. Both systems are known to crystallize very well. We are thoughtfully incorporating UAAs now we can see what they can do…We could also use these systems to vibrational probes into other (possibly more relevant systems). SHORTEN TITLE
- CO, O2, NO all bind Fe2+ (ferrous) form. Emphasize it is a protein class. Why H-NOX? Well defined expression/purification protocols, crystallizes readily, colored, blood substitute (omniox).
- Move earlier in talk to emphasize our motivation in choosing Y140 and how it will alter function. Mention specific UAAs that fit spectrum. Emphasize that these are expectations. Formal charges. Which is bigger? I or CN? O more polarizable, measurable pKa in titratable range. Amino group holds onto H more strongly, thus making it unavailable for an H bond.
- Personalize figure. Make everything explicit and easy to understand. Shrink plasmids. Make sure it is known plasmid is simply a piece of DNA.
- Separate gels on each slide with structures of UAA side chains below.
- More informative slide title. “Incorporating UAA….Something like that” A,B – traces surface around Van der Waals radii of all atoms in protein but not heme. White = nonempty space. Envelope around the heme, but not much room anywhere else. Show atoms/residues within 4-5 A in sticks. NOT necessary. Show beta carbon. Fix formal charges. Be consistent with lone pairs. Van der Waal radii of CN: 2.19 A? I: 1.98 – 2.15 A
- Change lighting on figure. Don’t clip off heme. Change clipping planes. Use + with far clip. Rendering – no shadows. Also look at residues closer to meta position. Move right image to the left and on the right include an the same iage but hide the phenyl ring…
- Be specific in mentioning motivation in choosing these sites. These sites are buried, solvent accessible, in O2 channel. Really just want to incorporate UAA and get crystals, but these sites still have meaning. Which sites are responsible for appropriate protein folding and function.
- Walk through structure. Show in surface? Play around with figure. These past slides shouldn’t have “Future directions”….but rather goals for next semester. Finish with an actual “Future Directions slide” Also, Acknowledgements slide. 4 things T4 allows us to study: 1. a new system to test vibrational probe reporter UAAs 2. structural stability 3. enzyme activity 4. slow down aggregation kinetics.
- Research corp. grant. People first, then other stuff. NSF info. Dr.