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150426-490 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. Overview Review System of Interest: Tt H-NOX In vivo incorporation of UAAs Steric hypothesis of UAA incorporation into heme pocket of Tt H-NOX New Data Tt H-NOX Y140TAG-F78A expressions with UAAs Tt H-NOX TAG mutant expressions with pCNF Future Directions Probe Tt H-NOX protein environment Tune O2 binding affinity
- 3. 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
- 4. In vivo Incorporation of UAAs Minnihan EC, Yokoyama K, Stubbe J - F1000 Biol Rep (2009) Incorporation machinery: tRNA and synthetase on same plasmid Gene with TAG site: on standard high-copy plasmid tRNA synthetase X H3N O O X H3N O O X H3N O O Ribosome tRNA tRNA with UAA Protein with UAA UAG 3'5' mRNA
- 5. 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 O2 affinity
- 6. SDS-PAGE gel of Tt H-NOX with UAAs L-4-Aminophenylalanine (pNH2F) L-3-Nitrotyrosine (mNO2Y) N H H HO N O O
- 7. SDS-PAGE gel of Tt H-NOX with UAAs L-4-Aminophenylalanine (pNH2F) L-3-Nitrotyrosine (mNO2Y) N H H HO N O O No incorporation of either pNH2F or mNO2Y
- 8. 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
- 9. SDS-PAGE gel of Tt H-NOX with UAAs Successful incorporation of halo-F’s but not pCNF Expression level: pClF > pBrF > pIF > pCNF STERICS? van der Waals radii (A) Cl: 1.77 Br: 1.92 I: 2.06 CN: 2.19A. Bondi (1964). "van der Waals Volumes and Radii". J. Phys. Chem. 68: 441
- 10. Goals for the Semester 1) Test the steric hypothesis of UAA incorporation into heme pocket of Tt H-NOX 2) Show that UAAs can be incorporated at various sites in Tt H-NOX Further mutate heme pocket of Tt H-NOX Choose sites of varying protein environments Incorporate pCNF at those sites Create TAG-mutants
- 11. Further Mutate the Heme Pocket PDB ID: 1U55 Y140 F78 OH O NH2 OH O NH2 SDM F78A Y140 A78 Phenylalanine Alanine
- 12. SDS-PAGE gel of Tt H-NOX with UAAs Repeated expression profile with the TtY140-F78A construct 20 25 50 kDa (+) P S P S P S Wt_H6 pNH2F mNO2Y P S P S P S pCNF pIF pBrF P S pClF pCNF pIF pBrF pClF ClBrIC N N H H pNH2F HO N O O mNO2Y
- 13. SDS-PAGE gel of Tt H-NOX with UAAs Repeated expression profile with the TtY140-F78A construct 20 25 50 kDa (+) P S P S P S Wt_H6 pNH2F mNO2Y P S P S P S pCNF pIF pBrF P S pClF Not quite… need to follow up with mass spec Successful incorporation of all UAAs – steric hypothesis confirmed?
- 14. Spectroscopic UAAs - pCNF Requirements of a spectroscopic reporter of protein environments Readily measurable spectroscopic observable C N ~ 2250 cm-1 ε = ~ 200 M-1cm-1 Observable is sensitive to local environment H2O THF ~ 8 cm-1 Incorporated into protein Stable Minimally invasive
- 15. Residue Total Apolar Backbone Sidechain Ratio In/out Y17 32.67 18.13 23.77 8.90 4.6 in F52 1.34 1.34 0.00 1.34 0.7 in F78 34.66 34.66 0.00 34.66 19.2 in F82 6.52 6.52 1.34 5.17 2.9 in Y85 27.54 25.75 0.38 27.16 14.1 in F86 16.48 11.03 5.44 11.03 6.1 in F94 7.55 7.55 0.00 7.55 4.2 in Y131 7.31 6.38 0.00 7.31 3.8 in Y138 19.25 18.28 0.55 18.70 9.7 in Y140 19.42 19.18 0.00 19.42 10.1 in F141 9.41 9.40 1.82 7.59 4.2 in F151 81.78 71.86 18.02 63.76 35.4 F152 28.71 16.21 18.12 10.60 5.9 in F169 84.71 84.64 0.14 84.58 47.0 F178 5.33 4.90 0.51 4.83 2.7 in F183 54.81 50.40 12.52 42.29 23.5 Y185 184.83 133.69 26.98 157.84 81.7 out Choosing Sites for pCNF Incorporation Highlighted residues represent a variety of environments in Tt H-NOX Buried sites: F52, F78, Y85 Partially buried sites: F151, F169, F183 Solvent exposed sites: Y185
- 16. Probe Other Sites in Tt H-NOX Incorporate pCNF at different sites Assess local protein environments with pCNF PDB ID: 1U55 Buried sites: F52, F78, Y85 Partially buried sites: F151, F169, F183 Solvent exposed sites: Y185
- 17. kDa (+) P S P S P S P S P S P S P S F52 F78 Y85 F151 F169 F183 Y185 50 25 20 SDS-PAGE gel of Tt H-NOX with pCNF L-4-Cyanophenylalanine pCNF C N
- 18. kDa (+) P S P S P S P S P S P S P S F52 F78 Y85 F151 F169 F183 Y185 50 25 20 SDS-PAGE gel of Tt H-NOX with pCNF L-4-Cyanophenylalanine C N We are able to incorporate pCNF at the sites of interest
- 19. Future Directions Tune the O2 binding affinity of Tt-HNOX by incorporating UAAs at the Y140 site Assess Tt H-NOX protein environment via pCNF 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 L-4-Cyanophenylalanine pCNF C N Using F78A constructs Using His6 constructs
- 20. 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 (22529) to C.M.PP. Franklin & Marshall College Department of Chemistry NSF (CHE-1053946) to S.H.B.
Editor's Notes
- Before I get started, I want to give a brief overview of my talk. I’ll start by reviewing where we left of at the end of the fall semester (reference sub bullets on slide), transition into I the new data we collected this semester (reference sub bullets on slide), and leave you with what we plan to do this summer and in the future (reference sub bullets).
- All our work has been completed in the Tt H-NOX system, which is a protein class containing the heme nitric oxide and oxygen binding (H-NOX) domain from the obligate anaerobe Thermoanaerobacter tencongensis (Tt H-NOX). Our motivation for using this system. Notably, there are well-defined expression/purification protocols, the protein crystallizes readily, the protein is red in color which aids in purification, and it is being developed as a blood substitute due to its affinity for oxygen (omniox). Shown below is the crystal structure of Tt H-NOX. Notable structural features of this protein are as follows: Heme containing – In ferrous form, the protein binds the three diatomic gas ligands shown (CO, O2, and NO). However, it should be mentioned that only those H-NOX domains containing a distal pocket Y are capable for binding O2. Lastly, I have the hydrogen binding environment shown here, whereby O2, which is bound above the heme is stabilized by an H-bond to the phenolic proton of Y140, which is subsequently stabilized by W9 and N74. Mutational studies have indicated the importance of Y140 for binding O2, whereby without Y140 Tt H-NOX has no affinity for O2. These analyses provide the basis for further motivation in using this system. Notably, Y140 has been shown to be crucial both for ligand discrimination and affinity and thus serves as a perfect candidate for UAA incorporation.
- The genetic incorporation of UAAs into proteins requires the use of two plasmids. The first of which contains a TAG-mutant of our gene of interest (in our case Tt H-NOX). What this means is that the site we wish to incorporate the UAA needs to be mutated to the nonsense TAG Amber codon. The second plasmid contains both thegenes for the tRNA with the anticodon for TAG, as well as the genes for the tRNA synthetase enzyme, responsible for covalently adding the UAA to the tRNA. We transform these two plasmids into competent E. coli cells and allow them to produce the protein for us. In essence, the tRNA synthetase charges the tRNA by covalently adding the UAA to the tRNA. This tRNA then binds to the complementary UAG site on the proteins mRNA, and then the bacterias endogenous translation material produces our protein with the UAA incorporated upon induction.
- To reiterate, Y140 in Tt H-NOX serves as a perfect candidate for UAA incorporation because that residue has been shown to be crucial for ligand discrimination and affinity. Thus, we can modulate O2 binding affinity by incorporating UAAs with different hydrogen bonding donatability. Shown here are two commercially available UAAs that allow us to modulate O2 binding in Tt H-NOX. On the left we have mNO2Y, which due to its withdrawing character allows it to be a stronger H-bond donor. And on the right we have pNH2F, which due to its stronger donating character, makes it a weaker H-bond donor…(these are expectations…O more polarizable, measurable pKa in titratable range. Amino group holds onto H more strongly, thus making it unavailable for an H bond).
- As mentioned, due to their utility in modulating Tt H-NOX O2 binding, we sought to express the mNO2Y and pNH2F constructs first. This SDS-PAGE protein gel, and those that will follow, are all set up as follows. Lane 1 is a protein ladder, with known molecular weight standards for comparison. Lane 2 is previously expressed and purified WT Tt H-NOX, and serves as a positive control and appears ~ 22 kDa. The lanes to follows show P and S lanes, which refer to pellet and supernatant following cell lysis for each of the labeled constructs. Because Tt H-NOS is a soluble protein, if the UAA was incorporated, and thus the protein produced, we expect to see a band in the S lane parallel to that of the positive control.
- The gel shows that both pNH2F and mNO2Y were not expressed in the Tt H-NOX-Y140 construct. Moreover, the Wt Tt_H6 was used for confirmation that it is failure of UAA incorporation that caused the expression to fail, and not details of the expression itself.
- Due to our failure to incorporate the larger pNH2F and mNO2Y UAAS, we attempted to incorporate other commercially available UAAs – pCNF, pClF, pBrF, and pIF. Thus, this SDS-PAGE protein gel is showing the same as the previous gel, except with different UAA containing constructs.
- This gel shows that the halogenated phenylalanine residues can be incorporated but that pCNF cannot. Further, it appears as though pClF expressed more than pBrF which expressed more than pIF, which when considering the van der Waal radii of these para-constituents lead to our consideration of sterics. (Measured (?) Van der Waal radii of CN: 2.19 and of I: 1.98 – 2.15 A).
- So that’s pretty much where we ended last semester, but moving forward into this semester our goals were two-fold. First, we wanted to test the steric hypothesis of UAA incorporation into the heme pocket of Tt H-NOX, which requires further mutations to be made in the heme pocket. Second, we wanted to show that UAAs can be incorporated at various sites, toward probing Tt H-NOX’s local protein environment with pCNF. This work includes choosing sites of varying protein environments, creating TAG mutants for those sites, and then incorporating pCNF at those sites.
- As I just mentioned, our first goal of the semester was to test this steric hypothesis. This image takes a deeper look at the heme pocket (in particular the residues close to Y140). It is clear that neighboring residue F78 imposes steric bulk to Y140, especially at the para-position. Thus, it stands to reason that removing this bulk, for example by mutating F78 to A78, we can incorporate larger UAAs. Ultimately, this will allow us to retackle the modulating O2 binding by UAA incorporation story.
- This SDS-PAGE protein gel shows the repeated expression profiles shown previously but with the new F78A constructs.
- The gel shows that we were able to successfully incorporate all UAAs tested. Thus, the F78A mutation was capable of facilitating the incorporation of larger UAAs.
- Nitrile symmetric stretch ~ 2250 cm-1. ε = 200 M-1cm-1 as compared to C-H ~ 1 M-1cm-1 and C=O (considered the ideal case) ~ 1200 M-1cm-1 (also, azide ~ 600-800 M-1cm-1.
- GETAREA data showing all native Y and F residues in Tt H-NOX. Highlighted residues indicate those that we wish to proceed with for candidate sites for pCNF incorporation to probe local protein environment. These sites span buried, partially buried, and solvent accessible environments.
- Using pCNF allows us to utilize the nitrile symmetric stretch vibration of the two-atom nitrile group. This vibrational mode result in a relatively intense IR absorbance band (compared with the Amide I transition) that is sensitive to local protein environments and appears in a relatively clear region of the IR spectrum. Another vobrational reporter UAA is pN3F, which has a higher peak extinction coefficient and greater sensitivity to local environment than the nitrile oscillator, suggesting that pN3F is the preferred vibrational UAA reporter. However, the azide group represents a greater possible structural perturbation than the nitrile group, and pN3F has been shown to be photoreactive. Thus, we will focus on enhancing the utility of the less invasive and more stable vibrational reporter, pCNF, which can be incorporated genetically into proteins with site specificity to serve as a sensitive probe of the local protein environment.
- Shows that we can indeed incorporate the UAA of interest (pCNF) at the sites needed for the vibrational probe work. Mention that F169’s tube spilt in the centrifuge, rather than it simply did not express
- Emphasize the main focuses for the summer. 1) Assuming the expression gel I am making now shows we indeed can express pNH2 and mNO2Y, we need to perform larger scales expressions and purifications of these constructs so that we can assess if O2 binding has been altered in response to UAA incorporation. 2) Probe the protein environment of Tt H-NOX with pCNF. At this point we have shown we can indeed express these constructs. Now, we simply need to perform larger scale expressions and purifications (Ni affinity chromatography) so that we can run protein IR.