KDM5 epigenetic modifiers as a focus for drug discovery
Tti2 Thesis Final Apr 8
1.
2. Elucidating the Structure and
Function of Protein Tti2
:: Selwyn Chui
:: Dr. Christopher J Brandl Lab
April 8, 2015::
Department of Biochemistry
3. :: Introduction
Tti2 is an essential yeast protein
• Tti2 = Tel2 (TELO2 in humans) Interacting Protein 2
o 421 amino acids (~49 kDa)
o Predicted to be mainly α-helical + chaperone
o ASTRA (Assembly of Tel, Rvb and Atm-like kinase) complex
o Triple T (TTT) complex: Tti1/2 and Tel2 (Telomere
maintenance protein 2)
Structure and Function of Tti2
4. Structure and Function of Tti2
:: Introduction
The TTT complex and PIKKs:
• TTT complex (along with co-chaperone Hsp90) helps with
folding/stabilization of PIKKs
PIKKS (Phosphatidylinositol 3-kinase-related protein kinases) are involved with
several essential cellular processes: DDR, metabolism, transcription, etc…
5. Structure and Function of Tti2
:: Introduction
The TTT complex and PIKKs:
• TTT complex (along with co-chaperone Hsp90) helps with
folding/stabilization of PIKKs
PIKKS (Phosphatidylinositol 3-kinase-related protein kinases) are involved with
several essential cellular processes: DDR, metabolism, transcription, etc…
6. :: Introduction
The TTT complex and PIKKs:
• TTT complex (along with co-chaperone Hsp90) helps with
folding/stabilization of PIKKs
PIKKS (Phosphatidylinositol 3-kinase-related protein kinases) are involved with
several essential cellular processes: DDR, metabolism, transcription, etc…
Structure and Function of Tti2
TTT complex helps
these proteins
function, but what
role does Tti2
play?
7. :: Introduction
Tti2 in other organisms
• Tti2 studied in yeast, but conserved in fungi and mammals;
a human Tti2 homolog also exists
Structure and Function of Tti2
8. :: Introduction
Tti2 in other organisms
• Tti2 studied in yeast, but conserved in fungi and mammals;
a human Tti2 homolog also exists
• Implicated in
human brain
development
Structure and Function of Tti2
9. :: Goal
The ultimate goal is to elucidate Tti2
structure, function, and protein interactions.
To this end, we want to identify alleles for
suppressor analysis.
Structure and Function of Tti2
10. :: Approach
Need to characterize Tti2 – How?
• Genetics approach: Altering individual Tti2 residues – are
they important for yeast growth in stress conditions? If yes,
we can proceed with…
• Suppressor analysis: Finding functionally related
proteins by selecting for suppressors of mutant
phenotypes
Eg. Tra1 (PIKK) functionally linked to Tti2to Y
But how do we pick residues to investigate?
Structure and Function of Tti2
12. :: Approach
Unigenic evolution analysis highlights
candidate key Tti2 residues
Some of these
residues were also
conserved between
Tti2 homologs…
Structure and Function of Tti2
L187: α-helix?
P228/P229: Random coil / turn?
Try P228G/P229A
13. :: Approach
Unigenic evolution analysis highlights
candidate key Tti2 residues
Some of these
residues were also
conserved between
Tti2 homologs…
Structure and Function of Tti2
(L187P tested…
Resists azetidine-
2-carboxylic acid)
L187: α-helix?
Try L187R
P228/P229: Random coil / turn?
Try P228G/P229A
14. :: Approach
Engineering mutant vectors:
1. Choose Tti2 alleles (L187R, P228G-P229A)
2. Site-directed mutagenic PCR of Tti2 gene
3. Digest & ligation into plasmid vector
4. Cloning in E. coli
• Plasmid amplification
Structure and Function of Tti2
Tti2-mutant,
LEU2, ampr
plasmid
15. :: Approach
Plasmid Insertion into Yeast
5. Plasmid purification from E. coli
6. Plasmid insertion into (ΔTti2 ΔLEU2 ΔURA3)
S. cerevisiae with URA3 Tti2WT plasmid
7. Plasmid Shuffling (ura+ yeast inviable
on 5-fluoroorotic acid)
Structure and Function of Tti2
5-FOA,
counterselects
ura+ cells
Tti2-wt, URA3 plasmid
Tti2-mutant, LEU2, ampr plasmid
Genome: ΔTti2 ΔLEU2 ΔURA3
16. :: Approach
Stress-Inducing Conditions
8. Observe yeast growth in response to stress
Tti2 stabilizes Tra1 (PIKK)
Crippled Tra1 grows slowly on ethanol / calcofluor white
Thus mutant Tti2 may also grow slowly on those media
• Ethanol
• Calcofluor white
Structure and Function of Tti2
17. :: Approach
Stress-Inducing Conditions
8. Observe yeast growth in response to stress
Tti2 stabilizes Tra1 (PIKK)
Crippled Tra1 grows slowly on ethanol / calcofluor white
Thus mutant Tti2 may also grow slowly on those media
• Ethanol
• Calcofluor white
L187P resists slow growth on azetidine
L187R may also grow differently
• Azetidine-2-carboxylic acid
• Heat Shock (37oC) **
Structure and Function of Tti2
18. ::Results
Tti2L187R and Tti2P228G-P229A: Ethanol
• Ethanol is a mild denaturant + growth inhibitor
Suggests leu/pro’s not vital for Tti2 response to ethanol
Structure and Function of Tti2
19. ::Results
Tti2L187R and Tti2P228G-P229A: Calcofluor White
• Calcofluor white (dye) binds the cell wall
Suggests leu/pro not vital for cell wall integrity
Structure and Function of Tti2
20. ::Results
Tti2L187R and Tti2P228G-P229A: Azetidine
• Azetidine-2-carboxylic acid toxic proline homologue
(WT sensitive to azetidine, but L187P conferred resistance; L187R
and prolines both same as WT)
Structure and Function of Tti2
21. ::Results
Tti2L187R and Tti2P228G-P229A: Heat shock
• Slow growth by increasing heat shock response
Suggests leu/pro not important for heat shock repsonse
Structure and Function of Tti2
22. ::Results
Structure and Function of Tti2
Structural Interpretation of Results
Pro228/229 Ala, Gly (More rotational freedom)
Prolines not functionally important?
Perhaps a linker region, not active site
23. ::Results
Structure and Function of Tti2
Structural Interpretation of Results
Pro228/229 Ala, Gly (More rotational freedom)
Prolines not functionally important?
Perhaps a linker region, not active site
Leu187 (NP) Arg (+)
But Leu187Pro had big effect?
Sidechain identity vs backbone structure
Not active site + Alpha helix disruption?
24. ::Results
Structure and Function of Tti2
Structural Interpretation of Results
Pro228/229 Ala, Gly (More rotational freedom)
Prolines not functionally important?
Perhaps a linker region, not active site
Leu187 (NP) Arg (+)
But Leu187Pro had big effect?
Sidechain identity vs backbone structure
Not active site + Alpha helix disruption?
Without a non-WT growth phenotype, we can’t do
suppressor analysis. Try a more severe allele?
25. ::Results
Combining proline and R1 region mutations:
Structure and Function of Tti2
Previously tested the (R1 region mutant)
Tti2W161A-I162A-P164A allele: Seemed to
have a slight effect (Data not published)
Will a combined allele give slower
growth?
26. ::Results
Combining proline and R1 region mutations:
Structure and Function of Tti2
Previously tested the (R1 region mutant)
Tti2W161A-I162A-P164A allele: Seemed to
have a slight effect (Data not published)
Will a combined allele give slower
growth?
…So severe, it’s not viable!
No Tti2
Tti2-WT
Tti2 mutant
W161A-I162A-P164A-
P228G-P229A
27. ::Results
Combining proline and R1 region mutations:
Structure and Function of Tti2
Previously tested the (R1 region mutant)
Tti2W161A-I162A-P164A allele: Seemed to
have a slight effect (Data not published)
Will a combined allele give slower
growth?
…So severe, it’s not viable!
What next??
No Tti2
Tti2-WT
Tti2 mutant
W161A-I162A-P164A-
P228G-P229A
28. ::Future Directions
Further test R1 region / proline residues
• Try lowering expression of Tti2
mutants for slow-growth phenotype
Continue investigating alleles
• Pick other hypomutable residues from
unigenic evolution analysis
• Trying different combinations of alleles
(Eg. L187R and the R1 region mutant)
Structure and Function of Tti2
29. ::Acknowledgements
Thanks to:
Dr. Christopher J Brandl, Julie Generaux,
Kyle Hoffman and Matthew Berg.
Also to Western University for providing the
opportunity and the facilities.
Questions?
Structure and Function of Tti2
30.
31. ::References
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2. Hurov KE, Cotta-Ramusino C and Elledge SJ (2010). A genetic screen identifies the Triple T complex required for DNA damage signalling and ATM and ATR stability. Genes Dev, 24(17):
1939-1950.
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4. Shevchenko A, Roguev A, Schaft D, Buchanan L, Habermann B, Sakalar C, Thomas H, Korgan NJ, Shevchenko A and Stewart AF (2008). Chromatin Central: towards the comparative
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5. Pal M, Morgan M, Phelps SEL, Roe SM, Parry-Morris S, Downs JA, PolierSigrun, Pearl LH and Prodromou C (2014). Structural basis for phosphorylation-dependent recruitment of Tel2 to
Hsp90 by Pih1. Structure, 22(6): 805-818.
6. Lee JH and Paull TT (2007). Activation and regulation of ATM kinase activity in response to double-strand breaks. Oncogene, 26(56): 7741-7748.
7.Khoronenkova SV and Dianov GL (2015). ATM prevents DSB formation by coordinating SSB repair and cell cycle regression. Proc Nalt Acad Sci U S A, pii: 201416031 [Epub ahead of print].
8. Zou L and Elledge SJ (2003). Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science, 300(5625): 1542-1548.
9. Hay N and Sonenberg N (2004). Upstream and downstream of mTOR. Genes Dev, 18(16): 1926-1945.
10. Runge KW and Zakian VA (1996). TEL2, an essential gene required for telomere length regulation and telomere position effect in Sacchormyces cerevisiae. Mol Cell Biol, 16(6): 3094-3105.
11. Kota RS and Runge KW (1998). The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA. Nucleic Acids Res, 26(6): 1528-1535.
12. Stirling PC, Bloom MS, Solanki-Patil T, Smith S, Sipahimalani P, Li Z, Kofoed M, Ben-Aroya S, Myung K and Hieter P (2011). The complete spectrum of yeast chromosome instability genes
identifies candidate CIN cancer genes and functional roles for ASTRA complex components.
13. Generaux J, Kvas S, Dobransky D, Karagiannis J, Gloor GB and Brandl CJ (2012). Genetic evidence links the ASTRA protein chaperone component Tti2 to the SAGA transcription factor Tra1.
Genetics, 191(3): 765-800.
14. Langouët M, Saadi A, Rieunier G, Moutton S, Siquier-Pernet K, FernetM,Nitschke P, Munnich A, Stern MH, Chaouch M and Colleaux L (2013). Mutation in TTI2 reveals a role for triple T
complex in human brain development. Hum Mutat, 34(11): 1472-1476.
15. Deminoff SJ, Tornow J and Santangelo GM (1995). Unigenic evolution: a novel genetic method localizes putative leucine zipper that mediates dimerization of the Saccharomyces
cerevisiae regulator Gcr1p. Genetics, 141(4): 1263-1274.
16. Boeke JD, Trueheart J, Natsoulis G and Fink GR (1987). 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol, 154: 164-175.
17. Saccharomyces Genome Database. http://www.yeastgenome.org/locus/S000003897/overview. SGD ID: YJR136C. Accessed Dec 20, 2014
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raghava/apssp2/. Accessed Sept 29, 2014.
Structure and Function of Tti2
Editor's Notes
Hi everyone, my name is Selwyn from the Brandl lab, and my thesis project focuses on elucidating the structure and function a protein called Tti2.
Tti2, or Tel2 interacting protein 2, is an essential yeast protein that’s thought to be a potential chaperone. It’s 421 amino acids long, approximately ~49 kDa in size, and is predicted to be mainly alpha-helical in structure. Tti2 is part of a few different complexes such as the ASTRA complex, whose function is not well characterized, and triple T complex, which is more well-studied. It’s composed of Tel2 as well as both Tti1 and 2.
So the triple T complex, along with heat shock protein HSP90, helps fold or stabilize a class of proteins called PIKKs (or phosphatidylinositol 3-kinase-related protein kinases). These related proteins share many conserved domains, such as the FAT and FATC domains; they’re involved in a wide variety of essential cellular processes. For instance, ATM and ATR initiate the DNA damage response pathway, and mTOR is a nutrient sensor that controls cell metabolism and growth.
Tra1 lacks kinase activity, unlike other PIKK family proteins (Human homolog is TRRAP)
- Targets SAGA and NuA4 to promoters; but also does other things (Helmlinger, 2011)
ATM = Ataxia-telangiectasia mutated portein (pp-lates key initiators of DNA repair, cell cycle arrest or apoptosis)
ATR = Ataxia-telangiectasia and Rad3 related protein (senses DNA damage, from radiation for example, and starts cell cycle arrest)
PIKKs have FATC domain; recruits Tip60 for histone acetylation
Tra1 is another PIKK that helps initiates transcription via histone deacetylase complexes. Tra1 in particular has been shown to be functionally related to Tti2, and the importance of this will become evident when I discuss my approach. These are all very important processes, which is part of the reason why…
Tra1 lacks kinase activity, unlike other PIKK family proteins (Human homolog is TRRAP)
- Targets SAGA and NuA4 to promoters; but also does other things (Helmlinger, 2011)
ATM = Ataxia-telangiectasia mutated portein (pp-lates key initiators of DNA repair, cell cycle arrest or apoptosis)
ATR = Ataxia-telangiectasia and Rad3 related protein (senses DNA damage, from radiation for example, and starts cell cycle arrest)
PIKKs have FATC domain; recruits Tip60 for histone acetylation
(part of the reason why)… it’s important to study Tti2. As part of the triple T complex, it helps maintain so many essential pathways, yet not much is known about it – we don’t have solved crystal structure and we don’t know many specifics about its protein-protein interactions. Additionally…
Tra1 lacks kinase activity, unlike other PIKK family proteins (Human homolog is TRRAP)
- Targets SAGA and NuA4 to promoters; but also does other things (Helmlinger, 2011)
ATM = Ataxia-telangiectasia mutated portein (pp-lates key initiators of DNA repair, cell cycle arrest or apoptosis)
ATR = Ataxia-telangiectasia and Rad3 related protein (senses DNA damage, from radiation for example, and starts cell cycle arrest)
PIKKs have FATC domain; recruits Tip60 for histone acetylation
Tti2 isn’t just yeast protein. It’s actually highly conserved in both fungi and mammals, (including humans!). In fact…
… there was a recent study done that implicated Tti2 in proper human brain development; where mutation in Tti2 led to an autosomal recessive condition. For these reasons, our ultimate goal is…
Now the question is, how do we go about doing that?? I took a genetics approach, by mutating individual Tti2 residues to see if they were important for function in in terms of how well a yeast colony can grow in stressful conditions. Additionally, if I could generate a growth-phenotype (Eg. Slow-growing in ethanol), I could do suppressor analysis. Suppressor analysis allows us to find functionally related proteins, by selecting for mutants that suppress or counteract the observed phenotype. This technique is actually how a functional link was found between Tti2 and the PIKK, Tra1 that I highlighted earlier.
Now, this is the sort of directrion I wanted to take my project towards, but the questions remains – how do I know which of the 421 residues to investigate?
The answer is two-fold: First, unigenic evolution analysis was done previously in the lab, not by me. And basically, it’s a stochastic method of identifying important, hypomutable residues in any protein. And when this was done for Tti2, we found that…
we found that… … some of these hypomutable residues also happened to be conserved between fungal homologs, and those were the ones I picked to investigate. For instance, the prolines at 228 and 229 are hypomutable and conserved, and are predicted to be part of a random coil / turn. These prolines had never been tested, so we decided to pick a conservative “neutral” mutation to glycine and alanine to see if that had any effect on Tti2 function.
The leucine at 187 is another residue that was hypomutable and conserved, and is predicted to be part of an alpha helix….
(L187)…This residue in particular, we’ve tested by mutating it into a proline. It was observed that this allele of Tti2 imparted resistance to the growth-inhibiting effects of azetidine-2-carboxylic acid, which is a toxic proline homologue. So mutations into prolines are unique because they tend to alter the backbone structure in addition to the sidechain. Thus, we wanted to try mutating the leucine to an acidic arginine to see if changing just the side chain would also give an effect.
The protocol itself was fairly straightforward. After we chose the alleles for testing, I designed and ordered the primers for mutagenic PCR, to incorporate the mutations. Then the PCR products were ligated into a plasmid vector with some selective markers, to be cloned and amplified in E. coli.
From the E. coli, I miniprepped the DNA and inserted them into yeast without Tti2 in its genome, but instead on a plasmid along with a URA3 marker, which is a very key consideration. The reason is that Tti2 is essential for cell viability. So to generate our mutant yeast, we had to use a plasmid shuffling technique. By plating the yeast with both plasmids onto media containing 5-fluoroorotic acid (or 5-FOA), the yeast will lose its wild type Tti2 plasmid, because ura+ cells metabolize 5-FOA into a toxic compound.
Finally, once we have our mutants generated, we can test them in various stress-inducing conditions. There were a few factors we considered before choosing the conditions. In the case of ethanol and calcofluor white, these agents were based on the evidence that Tti2 stabilizes Tra1, and that yeast with crippled Tra1 grew slowly on media containing both agents. Thus, it made sense to test Tti2 mutants in those same conditions, and expect a similar slow-growth phenotype as those Tra1 mutants.
In regards to azetidine, we knew that wild type Tti2 yeast are sensitive, and that the leucine to proline mutation at residue 187 conferred resistance to its growth-inhibiting effects. Thus, we particularly wanted to test our leucine to arginine mutant on it as well. We also decided to test temperature sensitivity of our alleles, as a general test of protein stability.
In regards to azetidine, we knew that wild type Tti2 strains are sensitive to it, and that the leucine to proline mutation conferred resistance to its growth-inhibiting effects. Thus, we particularly wanted to test our leucine to arginine mutant on it as well, since it was part of the reason we picked this mutation in the first place!
We also decided to test temperature sensitivity of our alleles, since it is a simple test of Tti2 response to protein instability.
First, we tested our leucine and proline mutants on 6% ethanol. Ethanol is a mild denaturant that inhibits yeast growth, which those of you who brew or even drink beer are probably familiar with. Even in the figure, you can see that it took the yeast about twice as long to grow to a similar size on the ethanol compared to regular YPD. But if you compare the wild type Tti2 in the middle row and compare to the proline *point* and leucine *point* mutants, the colony sizes are not much different. This suggests that the leucine and proline residues may not contribute much to Tti2 function, in terms of response to the growth-inhibiting effects of ethanol.
The next stressor we tested was calcofluor white. Calcofluor white is actually a fluorescent dye, but it interferes with cell wall assembly slowing down growth. This dye was shown to be effective at identifying yeast mutants that are sensitive to cell wall changes.
Similarly to the ethanol, there is not much difference between the colony sizes of the wild type and mutant Tti2. This suggests that these residues are not vital for maintaining cell wall integrity.
Next, we tested our mutants on azetidine-2-carboxylic acid (or just azetidine for the sake of simplicity). Azetidine a toxic proline homologue that competes against proline for incorporation into proteins. It changes the structure of the proteins, causing destabilization and growth inhibition. Wild type Tti2 strains are very sensitive to azetidine, as you can see *point*, as are both of my mutants. This is an interesting finding, particularly in the case of the leucine, because its mutation to proline conferred resistance, whereas its mutation to arginine (in my case) had little if any effect. This suggests that the backbone of residue 187 plays a role in azetidine resistance, since sidechain identity does not seem to be the cause, in the case of my arginine mutant. My proline mutants also do not exhibit any effect, suggesting that those residues may not be important to Tti2 function in response to destabilizing effects of azetidine.
Why minimal media here, instead of regular YPD?
Heat shock is also a common way of destabilizing and denaturing proteins, in a way different way compared to azetidine. We wondered if these Tti2 residues could be involved in the heat shock response, so we decided to test yeast at an elevated temperature of 37 degrees. Although all the strains grew more slowly at 37 compared to 30, the mutants and wild type colony sizes look the same. This suggests that these residues may not be important in terms of the heat shock response either.
my leucine and proline mutants at an elevated temperature of 37 degrees… Most yeast enzymes work optimally at 30 degrees so growing at 37 degrees may induce a heat shock response that will slow growth… You can sort of see this between the panels; the colonies in the grown at 37 seem to be a bit smaller over the same incubation period, which is what you’d expect. However, compared to the wild type growth shown in the middle row, the mutant colony sizes are almost the same, and this suggests that these particular residues might not contribute much to Tti2 structure and stability.
With these findings, there are some things we can learn about the nature of the residues we tested -- For instance, the prolines @ 228/229 were mutated into alanine and glycine, which (esp. gly) have a lot more rotational freedom in its backbone compared to the rigid ring structure in prolines. Since there was little effect on growth in conditions of stress, it’s likely that this region of Tti2 is not an important active site of sorts -- perhaps the prolines were just part of a linker region that can tolerate the increase in flexibility given by the glycine and alanine. This interpretation agrees with the secondary structure predictions, where this region is thought to be a turn or random coil.
The leucine is an interesting case, since there is no effect when mutated from non-polar sidechain into a positively charged arginine, suggesting the side chain does not play a big role in Tti2 (so again, likely not an important active site). However, this same leucine was already shown to be affected by mutation into proline, which not only affects the side chain but also the backbone. This is possibly because the leucine is part of an alpha-helix; prolines have poor helix-forming propensities, due to kinking the backbone and missing an amide hydrogen for hydrogen-bonding – again, this explanation also agrees with secondary structure predictions.
So those are a few interpretations we can speculate about, but if you remember that my goal was to find a slow-growing phenotype, you’ll notice I don’t have one. So I couldn’t really do any suppressor analysis. But when I got these results, I still had time left, so we decided to try a more severe allele.
The leucine is an interesting case, since there isn’t effect when mutated from non-polar sidechain into a positively charged arginine, suggesting the side chain identity does not play a big role in Tti2 in terms of our stress conditions. So again, this region is likely not an important active site. However, this same leucine was already shown to be affected by mutation into proline, which not only affects the side chain but also the backbone. This is possibly because the leucine is part of an alpha-helix, since prolines have poor helix-forming propensities, due to kinking the backbone and missing an amide hydrogen for hydrogen-bonding – again, this explanation also agrees with secondary structure predictions.
So those are a few interpretations we can speculate about, but if you remember that my goal was to find a slow-growing phenotype, you’ll notice I don’t have one. So I couldn’t really do any suppressor analysis. But when I got these results, I still had time left, so we decided to try a more severe allele.
So those are a few interpretations we can speculate about, but if you remember that one aspect of my goal was to find a growth phenotype, you’ll notice I don’t have one. So I couldn’t really do any suppressor analysis. So, we decided to try generating more severe allele of Tti2.
On the left of the red box are what we like to call the R1 region mutations, where the tryptophan, isoleucine and proline were mutated to alanines around ~160. This allele was tested in the Brandl lab before, and was found to grow very very slightly slower in heat or in the presence of ethanol. We wanted to see if we could generate a clearer slow-growth phenotype, by perhaps combining it with my proline mutant. So I digested my plasmids and inserted it into the R1 mutant vector.
*This is where I ran into a small problem, where we accidentally picked out the wrong DNA vector… After the arduous process of cloning my plasmids, transforming the yeast, and sequencing the DNA, we realized that the vector I was using contained a truncated Tti2, so of course the yeast was not viable. But after, using the CORRECT vector…
!! It turns out, the combined mutations of the R1 region and my prolines were severe enough to generate inviable yeast, as you can see from the bottom two rows on 5-FOA. The lack of growth in the mutants seems to be the same as not having Tti2 at all. This is especially interesting because individually, the alleles had almost no effect, yet together they completely shut down the yeast; This could be a result of a couple of things; maybe the prolines and the R1 region residues interact physically, or perhaps a threshold reduced Tti2 activity was crossed.
it’s important to note that Tti2 tends to be quite resilient, as it can often take many residue substitutions without compromising function, suggesting it is not a delicate enzyme where a single mutation will break the whole thing, but perhaps as long as it’s general structure is maintained, it can function normally.
From this, we know that these R1 /+ prolines residues are somewhat important to Tti2 function – this gives us a direction to pursue in terms of finding alleles for suppressor analysis….
In our lab, we use a very strong promoter to express Tti2, since the natural promoter is too weak. One possibility is that our mutations actually are affecting Tti2 activity, but because there is such a high expression through the promoter, the total activity is not reduced enough to generate a slow-growth phenotype. So instead, we can replace the natural promoter to see if these lower expression versions of our alleles will grow differently; and if successful, we can do suppressor analysis on those mutants.
Otherwise, we can continue investigating new alleles, maybe by picking other hypomutable residues from unigenic evolution, or trying different combinations of alleles that we’ve already made. Hopefully, these next steps are able to uncover more clues about the structure and function of Tti2.
