This document summarizes research aiming to characterize the steric limitations of incorporating unnatural amino acids (UAAs) into the heme binding pocket of the gas-binding protein Tt H-NOX from Thermoanaerobacter tengcongensis. The researchers incorporated various UAAs at position 140 in the heme pocket and found that larger UAAs decreased protein expression levels. They also incorporated the UAA p-cyanophenylalanine (pCNF) at multiple sites in Tt H-NOX and observed incorporation at all sites except position 169. The goal is to tune the protein's oxygen binding affinity by introducing UAAs near the heme group.

1. Characterizing steric limitations of the heme pocket in the gas-binding
Tt H-NOX protein using site-specific incorporation of unnatural amino acids.
Lukasz T. Olenginski and Christine M. Phillips-Piro
Franklin & Marshall College, Department of Chemistry, Lancaster, PA 17604-3003
Increasing size of UAA at 140 site decreases level of expression
Figure 4. SDS-PAGE gel of various Tt H-NOX constructs containing UAAs at the 140 site following
first steps of purification. As indicated by lane 2, Tt H-NOX protein should appear ~ 22 kDa. Other
than the WT H_6 construct (lanes 7, 8), only the halogenated phenylalanine UAA containing
constructs expressed well (lanes 11-16). Further, expression levels decreased as the size of the
halogen constituent increased.
Incorporation of UAAs at various sites
References
(1) Pellicena, P., Karow, D. S., Boon, E. M., Marletta, M. A., and Kuriyan, J. (2004) Crystal structure of an oxygen-binding
heme domain related to soluble guanylate cyclases. Proc. Natl. Acad. Sci. 101, 12854-12859.
(2) Karow, D. S., Pan, D., Tran, R., Pellicena, P., Presley, A., Mathies, R. A., and Marletta, M.A. (2004) Spectroscopic
characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholera and Thermoanaerobacter
tengcongensis. Biochemistry. 43, 10203-10211.
(3) Derbyshire E. R., Deng S., and Marletta M. A. (2010) Incorporation of tyrosine and glutamine residues into the soluble
guanylate cyclase heme distal pocket alters NO and O2 binding. J Biol Chem. 285, 17471-8.
(4) Olea, C., Boon, E. M., Pellicena, P., Kuriyan, J., and Marletta, M. A. (2008) Probing the function of heme distortion in
the H-NOX family. ACS Chem. Biol. 3, 703-710.
(5) Miyake-Stoner, S. J., Miller, A. M., Hammill, J. T., Peeler, J. C., Hess, K. R., Mehl, R. A., and Brewer, S. H. (2009)
Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan.
Biochemistry
48, 5953–5962.
(6) Bazewicz, C. G., Lipkin, J. S., Smith, E. E., Liskov, M. T., and Brewer, S. H. (2012) Expanding the utility of 4-cyano-L-
phenylalanine as a vibrational reporter of protein environments. J Phys Chem B 116, 10824–10831.
Acknowledgements
- Hackman Scholar Program, Marshall Scholar fund to L.T.O., Franklin & Marshall College
- Dr. Scott H. Brewer, Gregory Olenginski, Elise Tookmanian, Nicole Maurici, Daniyal
Tariq, Lisa Mertzman, Julie Gemmell
Heme Nitric Oxide and/or Oxygen binding (H-NOX) proteins
Figure 1. The heme binding pocket of Tt H-NOX with O2 bound (PDB ID: 1U55). The heme and
some residues shown in orange sticks. Y140 and H-bonding network known to be crucial for tight
O2 binding affinity.
H102
W9
N74
Y140
In vivo incorporation of UAAS
Adapted from: Minnihan EC, Yokoyama K, Stubbe J - F1000 Biol Rep (2009)
OH
O
NH2
OH
O
NH2
SDM
F78A
Phenylalanine Alanine
Y140
F78
Y140
A78
Thermoanaerobacter tencongensis (Tt H-NOX)
Assess protein structure, stability, and function with different UAAs
- Research Corp. Grant (22529) to C.M.PP.
- NSF (CHE-1053946) to S.H.B
Figure 6. Tt H-NOX shown in cartoon representation (PDB ID: IU55) with select tyrosine and
phenylalanine residues shown in yellow sticks and colored by atom, indicating where TAG sites
have been added to allow for UAA incorporation.
Figure 5. Heme pocket of Tt H-NOX shown in cartoon (PDB ID: IU55). Y140 with both F78 (left)
and A78 (right) shown in spheres, highlighting steric constraints within the heme pocket.
Incorporation machinery:
tRNA and synthetase on same
plasmid
Gene with TAG site:
on standard high-copy plasmid
Unnatural Amino Acids (UAAs)
N
H
H
HO
N
O O
(1) (2) C
N (3)
Cl Br I
(4) (5) (6)
Figure 2. Unnatural amino acids (UAAs) used for incorporation at Y140 of Tt H-NOX. (1) L-4-
aminophenylalanine (pNH2F) (2) 3-nitro-tyrosine (mNO2Y) (3) L-4-cyanophenylalanine (pCNF)
(4) L-4-chlorophenylalanine (pClF) (5) L-4-bromophenylalanine (pBrF) (6) L-4-iodophenylalanine
(pIF).
Further mutate the heme pocket
Incorporation of UAAs at the 140 site
kDa (+) P S P S P S P S P S P S P S
50
25
20
pNH2 mNO2Y WT_H6 pCNF pClF pBrF pIF
Goals
- Characterize steric limitations to incorporating larger UAAs into the Tt H-NOX scaffold
- Tune the O2 binding affinity of Tt-HNOX by incorporating UAAs at the Y140 site
- Assess Tt-HNOX protein environment/stability via UAA incorporation
Figure 3. Impact of addition of ALA on Tt H-NOX_Y140 + pCNF expression. Parallel 250 ml
expressions were setup in 500 ml baffled flasks both with (left) and without (right) addition of ALA.
(A) Expression cultures after 30-36 hr growth period. (B) Supernatant after lysis and first steps of
purification.
Addition of δ-aminolevulinic acid (ALA) aids expression of Tt H-NOX
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
Figure 7. SDS-PAGE gel of Tt H-NOX TAG mutants + pCNF following first steps of
purification. As indicated by lane 2, Tt H-NOX protein should appear ~ 22 kDa. Successful
pCNF incorporation was seen at all of the sites (lanes 3-10, 13-16) except the F169 site (lanes
11, 12).
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
Incorporation of pCNF at various sites of Tt H-NOX
A B
H-NOX Proteins