Part:BBa_K2980011

CRY2(R489E)

CRY2(R489E) is a mutant of Cryptochrome 2 (CRY2). By changing arginine on 489 to glutamate, the charge of C terminal is changed and the ability of CRY2 homo-oligomerization is lowered.

Background

In our system, phases are formed by CIB1-GCN(4)-FUS-GFP, while the recruitment of downstream proteins fused with mCherry-CRY2 into the phase is presented via the interaction between mCherry-CRY2 and CIB1-GCN(4)-FUS-GFP when stimulated. Therefore, without stimulation of 488nm laser, we anticipate that mCherry-CRY2 is smear in E.coli. Once stimulated, with strong interaction of CRY2-CIB1, mCherry-CRY2 is pulled into the phase. However, CRY2 homo-oligomerization is also stimulated by light 488nm, observed in our control experiment, which might alter the function of our system.

Design

To solve the problem, we introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.

Mutants	Principle
CRY2(R489E)	Change arginine on 489 to glutamate, making charge of C terminal negative.
CRY2(R489D)	Change arginine on 489 to aspartate, making charge of C terminal negative.
CRY2(Δ489-498)	Delete amino acids from 489-498, making charge of C terminal negative.
Table 1. Mutants of CRY2

Result

After observing under confocal, we found the extent of homo-oligomerization of CRY2 (R489E) is the lowest. Figure 1 shows confocal images of CRY2wt and CRY2(R489E).

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  Figure 1A Confocal image of CRY2wt (Zoom 1), most E.coli form phases, showing high ability of oligomerization
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  Figure 1B Confocal image of CRY2 (R489E)(Zoom 1), mCherry in E.coli are smear, showing low abilityof oligomerization.

All samples are induced with 0.05mM IPTG for 2 h at 16℃. Figure 2 shows homo-oligomerization details of CRY2wt and CRY2(R489E).

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  Figure 2A Confocal image of CRY2wt(Zoom 3), CRY2wt forms 2 phases on both ends of E.coli
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  Figure 2B Confocal image of CRY2(R489E)(Zoom 3), CRY2(R489E) is almost smear in E.coli.

Although in some cells phase is formed, the phase is much more smaller and darker than the phase formed by CRY2wt. All samples are induced with 0.05mM IPTG for 2 h at 16℃ We carried out statistical analysis after acquiring merge images (mCherry channel and TD). Three kinds of objects are count in our observation: bacteria with phase separation, bacteria without phase separation, bacteria whose state was unclear (probably because of low expression level for unknown reason or too young to accumulate enough proteins). The examples of captured pictures were given in Figure 3. Then, the total number of bacteria was added up. Finally, we used the ratio of group “phase” to sum to indicate the ability of CRY2 oligomerization under the same condition. The distributions of the data are showed in Figure 4.

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  Figure 3A example of E.coli with phase separation.
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  Figure 3B example of E.coli without phase separation.
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  Figure 3C example of unclear situation E.coli.

Reference

[1] Yu, X., Liu, H., Klejnot, J., & Lin, C. (2010). The Cryptochrome Blue Light Receptors. The Arabidopsis Book, 8(8). doi:10.1199/tab.0135

[2] Engelhard, C., Wang, X., Robles, D., Moldt, J., Essen, L., Batschauer, A., ... & Ahmad, M. (2014). Cellular Metabolites Enhance the Light Sensitivity of Arabidopsis Cryptochrome through Alternate Electron Transfer Pathways. The Plant Cell, 26(11), 4519-4531.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 723
  Illegal PstI site found at 533
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 723
  Illegal PstI site found at 533
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 723
  Illegal BglII site found at 393
  Illegal BglII site found at 852
  Illegal BamHI site found at 1331
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 723
  Illegal PstI site found at 533
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 723
  Illegal PstI site found at 533
  Illegal AgeI site found at 277
  Illegal AgeI site found at 1006
  
• 1000
  COMPATIBLE WITH RFC[1000]