Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium
Abstract
:1. Introduction
2. Results
2.1. Prototrophic E. coli Grow in Mineral Medium Supplemented with pMet and Aha but Not Hpg
2.2. Prototrophic E. coli Undergo a Second Growth Phase, Whereas Auxotrophic E. coli Enter a Late Stationary Phase When Depleting Methionine Accumulated in Cells by Transfer from Nutritionally Rich to Mineral Medium with ncAA
2.3. Protein Expression Rates Are Higher in the Presence of pMet Than in the Presence of Aha and in Prototrophic Than in Auxotrophic E. coli
2.4. Incorporation of Non-Canonical Amino Acids
3. Discussion
4. Materials and Methods
4.1. Materials and Reagents
4.2. Bacterial Growth
4.3. Protein Expression
4.4. Electrophoresis and Protein Digestion
4.5. MALDI-TOF Analysis
4.6. LC-MS/MS Analysis
4.7. Estimation of the Frequency of Methionine Analog Incorporation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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E. coli BL-21 | E. coli B834 | |||||||
---|---|---|---|---|---|---|---|---|
Met | pMet | Aha | Hpg | Met | pMet | Aha | Hpg | |
r | 0.82 | 0.47 | 0.52 | - | 0.63 | - | - | - |
K | 1.79 | 2.00 | 1.97 | - | 1.80 | - | - | - |
t5%K [h] | 4.2 | 7.1 | 6.6 | - | 3.3 | - | 16.1 * | - |
t1%r [h] | 13.3 | 23.3 | 21.0 | - | 15.2 | - | >25.0 | - |
Protein | Methionine Sites in | Sequence | From-To | Monoisotopic Mass (Da) | |
---|---|---|---|---|---|
Protein | Peptide | ||||
b5M46 | 1 | 1 | K.FLEEHPMGEEVLR.E | 39–51 | 1584.7606 |
MBP-GFP | 12 | 1 | K.SAMPEGYIQER.T | 494–504 | 1279.5867 |
2 | K.GEIMPNIPQMSAFWYAVR.T | 327–344 | 2109.0176 | ||
pspA | 7 | 1 | R.QQALMLR.H | 136–142 | 858.4746 |
2 | R.LMIQEMEDTLVEVR.S | 31–44 | 1704.8426 |
Amino Acid | Side Chain | Formula | Monoisotopic Mass (Da) | Δ Mass (AA-Met) (Da) | Origin of the Derivative |
---|---|---|---|---|---|
Methionine | -(CH2)2-S-CH3 | C5H11NO2S | 149.0510 | - | - |
MetOx | -(CH2)2-S(=O)-CH3 | C5H11NO3S | 165.0460 | 15.9950 | sp |
pMet | -(CH2)2-C(=N2)-CH3 | C6H11N3O2 | 157.0851 | 8.0341 | - |
pMet D1 | -(CH2)2-CH=CH2 | C6H11NO2 | 129.0790 | −19.9720 | pi |
-CH2-CH=CH-CH3 | |||||
Aha | -(CH2)2-N=N+=N− | C4H8N4O2 | 144.0647 | −4.9863 | - |
Aha D1 | -(CH2)2-NH2 | C4H10N2O2 | 118.0742 | −30.9768 | bc/sp |
Aha D2 | -(CH2)2-OH | C4H9NO3 | 119.0582 | −29.9928 | bc/sp |
Aha D3 | -CH=CH2 | C4H7NO2 | 101.0477 | −48.0033 | bc/sp |
Hpg | -(CH2)2-C≡CH | C6H9NO2 | 127.0633 | −21.9877 | - |
ncAA | pMet (%) | Aha (%) | Hpg (%) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Approach | MS | LC-MS | MS | LC-MS | MS | LC-MS | ||||||
Peptide | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 |
Ave (P1/2) | 56.8 | 54.2 | 74.1 | 73.0 | 64.6 | 73.3 | 53.0 | 57.0 | 46.6 | 59.7 | 51.4 | 58.9 |
S.D. (P1/2) | 5.4 | 3.6 | 1.5 | 0.9 | 2.6 | 2.1 | 2.7 | 3.1 | 7.3 | 6.1 | 4.1 | 5.9 |
Ave (total) | 55.5 | 73.5 | 68.9 | 55.0 | 53.1 | 54.7 | ||||||
S.D. (total) | 4.8 | 1.3 | 5.0 | 3.5 | 9.4 | 6.2 | ||||||
Δ Ave (P1-P2) | 2.7 | 1.1 | −8.7 | −3.9 | −13.1 | −7.5 | ||||||
Δ Ave (MS-LCMS) | −17.3 | −18.8 | 11.6 | 16.4 | −4.8 | 0.8 |
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Jecmen, T.; Tuzhilkin, R.; Sulc, M. Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium. Int. J. Mol. Sci. 2023, 24, 11779. https://doi.org/10.3390/ijms241411779
Jecmen T, Tuzhilkin R, Sulc M. Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium. International Journal of Molecular Sciences. 2023; 24(14):11779. https://doi.org/10.3390/ijms241411779
Chicago/Turabian StyleJecmen, Tomas, Roman Tuzhilkin, and Miroslav Sulc. 2023. "Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium" International Journal of Molecular Sciences 24, no. 14: 11779. https://doi.org/10.3390/ijms241411779
APA StyleJecmen, T., Tuzhilkin, R., & Sulc, M. (2023). Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium. International Journal of Molecular Sciences, 24(14), 11779. https://doi.org/10.3390/ijms241411779