Naomi Abraham, Lewis Churchfield, Dr. F. Akif Tezcan · METAL BINDING AND DISULFIDE BOND BREAKAGE...
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L.A. Churfield, F. Medina-Morales, J.D. Brodin, A. Perez, F.A. Tezcan. De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds, J. Am. Chem. Soc. (2016) J.B. Bailey, R.H. Subramanian, L.A. Churchfield, F.A. Tezcan. Metal-Directed Design of Supramolecular Protein Assemblies, Meth. Enzymol. (2016). Abstract Results Part III (Gel + Protein treatments) Conclusions and Future Directions Proteins are molecules in cells that carry out many important tasks, including carrying oxygen in our blood, converting food to energy, and communicating information about a cell’s environment to the inside of a cell. Since proteins serve many functions we are interested in engineering proteins that have new properties. Previously, we engineered a protein, C38/C81/C96 R1, that forms a tetramer which binds Zn and which contains six disulfide bonds. Surprisingly, when the Zn is removed, one C38/C38 disulfide bond breaks. We are working with the C38 RIDC1 protein and hope to find that, unlike C38/C81/C96 R1, it can keep a disulfide bond when Zn is removed. In order to study this, we must purify our protein from E. coli cell culture. Then we can combine our protein with chelators, reductants, and our Zn metal to determine if C38 R1 can bind to zinc and create a disulfide bond without the disulfide bond formation being metal dependent. SDS-PAGE Gel References Acknowledgments Introduction & Background METAL BINDING AND DISULFIDE BOND BREAKAGE CAN BE DECOUPLED IN AN ENGINEERED METALLOPROTEIN Naomi Abraham, Lewis Churchfield, Dr. F. Akif Tezcan Results Part II (PAR Assay) Five sample treatments are shown in the gel above: 1. Isolated protein : The isolated protein is simply purified protein 2. Apo protein : protein is chelated then ran through a column to separate the protein from the metal- bound chelator 3. TCEP + Protein : TCEP is a reductant whose job is to show the breakage of the disulfide bond. The monomer band indicates that the sample without a reductant formed a disulfide bond. 4. Zn + Protein: the disulfide bond forms in the presence of Zn which acts like the C38/C81/C96 R1 5. Zn + IAA + EDTA + Protein: Adding IAA and EDTA to the Zn + protein sample shows that the disulfide bond does not break, despite the fact that chelators remove the metal. Lastly, IAA would capture any cysteines if the disulfide bond broke, which was not observed. The above results show the C38 R1 protein behaves differently than the C38/C81/C96 R1 protein because the C38 R1 protein gel only shows dimers, and not a mix of monomers and dimers. This indicates that the disulfide bond never breaks in the C38 R1 protein. Conclusions • The C38R1 protein can bind Zn and can create a disulfide bond • The disulfide bond is not dependent on Zn • The breakage of the C38/C38 disulfide in C38/C81/C96R1 is probably due to the protein environment and not a property of the disulfide bond by itself. Future Directions • Determine x-ray crystal structure C38R1 (apo and Zn-bound) • Repeat PAR assay to determine why Zn/protein was so low I would like to thank Dr. Elizabeth Komives, Lewis Churchfield, Dr. Akif Tezcan, and Academic Connections for providing me with the opportunity to conduct research in a lab. Results Part I ( C38 R1 Purification) 1. Add metal binding amino acids to make metal-dependent protein assemblies. 2. Add hydrophobic interfaces that allow protein to self assemble. 3. Add disulfide bonds can break or relax when the metal is removed. (1) (2) + − + − + − (3) Protein monomer (cyt cb 562 ) C38 R1 Cell Pellet Purified C38 R1 Zn does not absorb visible light, but we can bind it to PAR so that we can see the absorbance of the Zn- PAR complex which allows us to see the amount of Zn in a sample. For the Zn-Par Standard Curve we incrementally added Zn to our PAR solution. This gives us a way to convert between absorbance at 500 nm (A 500 ) and the concentration of Zn ([Zn]). The difference between our protein and par absorbance and PAR/EDTA/protein absorbance give us the absorbance of zinc was lower than expected at 0.12 Zn per protein. We expect that C38 R1 can bind 1 Zn per protein. 0 0.5 1 1.5 200 300 400 500 600 Absorbance (AU) Wavelength (nm) UV-Vis Absorption Spectra Crude Purified • Express protein in E. coli cell culture • Sonicate cell pellet • Run protein through CM-sepharose column (cation exchanger) in acetate (acid buffer) • Run protein through Q-column (anion exchanger) in phosphate (basic buffer) • Run through S75 column (size-exclusion) on the FPLC • Concentrated purified protein R² = 0.9952 0 0.2 0.4 0.6 0.8 1 1.2 0 10 20 Absorbance at 500 nm [Zn] (μM) Zn 2+ -PAR Standard Curve 2mer 1mer 0 1 2 3 4 300 500 700 Absorbance (AU) Wavelength (nm) PAR absorbance spectra No Zn + Zn Sample Zn/Protein Apo C38 R1 ~0 Zn- C38 R1 0.12 The previously studied C38/C81/C96 R1 contains six disulfide bonds, one of which, C38/C38, breaks when the Zn is removed from the tetramer. There are two possible reasons for this behavior: 1. The protein environment can affect whether the disulfide bond remains or breaks. 2. It is a fragile bond that will always break. We wanted to determine which possibility is true by studying C38 R1, which we expect can form a C38/C38 disulfide bond, and which has no other disulfide bonds. 1 2 3 4 5
Transcript of Naomi Abraham, Lewis Churchfield, Dr. F. Akif Tezcan · METAL BINDING AND DISULFIDE BOND BREAKAGE...
L.A. Churfield, F. Medina-Morales, J.D. Brodin, A. Perez, F.A. Tezcan. De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds, J. Am. Chem. Soc. (2016)
J.B. Bailey, R.H. Subramanian, L.A. Churchfield, F.A. Tezcan. Metal-Directed Design of Supramolecular Protein Assemblies, Meth. Enzymol. (2016).
Abstract Results Part III (Gel + Protein treatments)
Conclusions and Future Directions
Proteins are molecules in cells that carry out many important tasks, including carrying oxygen inour blood, converting food to energy, and communicating information about a cell’s environmentto the inside of a cell. Since proteins serve many functions we are interested in engineeringproteins that have new properties. Previously, we engineered a protein, C38/C81/C96R1, that forms atetramer which binds Zn and which contains six disulfide bonds. Surprisingly, when the Zn isremoved, one C38/C38 disulfide bond breaks.
We are working with the C38RIDC1 protein and hope to find that, unlike C38/C81/C96R1, it can keep adisulfide bond when Zn is removed. In order to study this, we must purify our protein from E. colicell culture. Then we can combine our protein with chelators, reductants, and our Zn metal todetermine if C38R1 can bind to zinc and create a disulfide bond without the disulfide bondformation being metal dependent.
SDS-PAGE Gel
References
Acknowledgments
Introduction & Background
METAL BINDING AND DISULFIDE BOND BREAKAGE CAN BE DECOUPLED
IN AN ENGINEERED METALLOPROTEINNaomi Abraham, Lewis Churchfield, Dr. F. Akif Tezcan
Results Part II (PAR Assay)
Five sample treatments are shown in the gel above: 1. Isolated protein: The isolated protein is simply purified protein2. Apo protein: protein is chelated then ran through a column to separate the protein from the metal-
bound chelator3. TCEP + Protein: TCEP is a reductant whose job is to show the breakage of the disulfide bond. The
monomer band indicates that the sample without a reductant formed a disulfide bond.4. Zn + Protein: the disulfide bond forms in the presence of Zn which acts like the C38/C81/C96R15. Zn + IAA + EDTA + Protein: Adding IAA and EDTA to the Zn + protein sample shows that the disulfide
bond does not break, despite the fact that chelators remove the metal. Lastly, IAA would capture any cysteines if the disulfide bond broke, which was not observed.
The above results show the C38R1 protein behaves differently than the C38/C81/C96R1 protein because theC38R1 protein gel only shows dimers, and not a mix of monomers and dimers. This indicates that thedisulfide bond never breaks in the C38R1 protein.
Conclusions• The C38R1 protein can bind Zn and can create a disulfide bond• The disulfide bond is not dependent on Zn • The breakage of the C38/C38 disulfide in C38/C81/C96R1 is probably due to the protein environment
and not a property of the disulfide bond by itself.
Future Directions• Determine x-ray crystal structure C38R1 (apo and Zn-bound)• Repeat PAR assay to determine why Zn/protein was so low
I would like to thank Dr. Elizabeth Komives, Lewis Churchfield, Dr. Akif Tezcan, and Academic Connections for providing me with the opportunity to conduct research in a lab.
Results Part I (C38R1 Purification)
1. Add metal binding amino acids to make metal-dependent protein assemblies.
2. Add hydrophobic interfaces that allow protein to self assemble.
3. Add disulfide bonds can break or relax when the metal is removed.
(1) (2)
+− +− +−
(3)
Protein monomer(cyt cb562)
C38R1 Cell Pellet
Purified C38R1
Zn does not absorb visible light, but we can bind it to PAR so that we can see the absorbance of the Zn-PAR complex which allows us to see the amount of Zn in a sample.
For the Zn-Par Standard Curve we incrementally added Zn to our PAR solution. This gives us a way to convert between absorbance at 500 nm (A500) and the concentration of Zn ([Zn]).
The difference between our protein and par absorbance and PAR/EDTA/protein absorbance give us the absorbance of zinc was lower than expected at 0.12 Zn per protein. We expect that C38R1 can bind 1 Zn per protein.
0
0.5
1
1.5
200 300 400 500 600
Ab
sorb
ance
(A
U)
Wavelength (nm)
UV-Vis Absorption Spectra
Crude
Purified
• Express protein in E. coli cell culture
• Sonicate cell pellet
• Run protein through CM-sepharose
column (cation exchanger) in acetate
(acid buffer)
• Run protein through Q-column (anion
exchanger) in phosphate (basic buffer)
• Run through S75 column (size-exclusion)
on the FPLC
• Concentrated purified protein
R² = 0.9952
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20
Ab
sorb
ance
at
50
0 n
m
[Zn] (µM)
Zn2+-PAR Standard Curve
2mer
1mer
0
1
2
3
4
300 500 700
Ab
sorb
ance
(A
U)
Wavelength (nm)
PAR absorbance spectra
No Zn+ Zn
Sample Zn/Protein
Apo C38R1 ~0
Zn-C38R1 0.12
The previously studied C38/C81/C96R1 contains six disulfide bonds, one of which, C38/C38, breakswhen the Zn is removed from the tetramer. There are two possible reasons for this behavior:
1. The protein environment can affect whether the disulfide bond remains or breaks.2. It is a fragile bond that will always break.
We wanted to determine which possibility is true by studying C38R1, which we expect can form aC38/C38 disulfide bond, and which has no other disulfide bonds.
1 2 3 4 5
