Nano differential scanning fluorimetry
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NanoDSF is a modified differential scanning fluorimetry method to determine protein stability employing intrinsic tryptophan or tyrosine fluorescence.[1][2]
Protein stability is typically addressed by thermal or chemical unfolding experiments.[3] In thermal unfolding experiments, a linear temperature ramp is applied to unfold proteins, whereas chemical unfolding experiments use chemical denaturants in increasing concentrations. The thermal stability of a protein is typically described by the 'melting temperature' or 'Tm', at which 50% of the protein population is unfolded, corresponding to the midpoint of the transition from folded to unfolded.
In contrast to conventional DSF methods, nanoDSF uses tryptophan or tyrosine fluorescence to monitor protein unfolding. Both the fluorescence intensity and the fluorescence maximum strongly depends on the close surroundings of the tryptophan.[4] Therefore, the ratio of the fluorescence intensities at 350 nm and 330 nm is suitable to detect any changes in protein structure, for example due to protein unfolding.
Its applications include antibody engineering, membrane protein research, quality control and formulation development.[5][6] NanoDSF has also been utilized to rapidly evaluate the melting points of enzyme libraries for biotechnological applications. [7]
Examples for application of nanoDSF
[edit]The nanoDSF technology was used to confirm on-target binding of BI-3231 to HSD17B13 and to elucidate its uncompetitive mode of inhibition with regards to NAD+.[8]
References
[edit]- ^ Käufl F, Rapp F. "nanoDSF". NanoTemper Technologies GmbH.
{{cite journal}}: Cite journal requires|journal=(help) - ^ "Protein Stability Measurement Instrument". Genetic Engineering & Biotechnology News. 35 (4). 15 February 2015. Archived from the original on 12 September 2015. Retrieved 9 March 2015.
- ^ Senisterra G, Chau I, Vedadi M (April 2012). "Thermal denaturation assays in chemical biology". Assay and Drug Development Technologies. 10 (2): 128–36. doi:10.1089/adt.2011.0390. PMID 22066913.
- ^ Lakowicz JR (2006). Principles of Fluorescence Spectroscopy (3rd ed.). Springer US. ISBN 978-0-387-31278-1.
- ^ Käufl F, Rapp F. "Application Notes: nanoDSF Technology". NanoTemper Technologies GmbH. Archived from the original on 2015-02-05. Retrieved 2015-03-02.
- ^ "App Notes". Unchained Labs.
- ^ Magnusson AO, Szekrenyi A, Joosten HJ, Finnigan J, Charnock S, Fessner WD (January 2019). "nanoDSF as screening tool for enzyme libraries and biotechnology development". The FEBS Journal. 286 (1): 184–204. doi:10.1111/febs.14696. PMC 7379660. PMID 30414312.
- ^ Thamm, Sven; Willwacher, Marina K.; Aspnes, Gary E.; Bretschneider, Tom; Brown, Nicholas F.; Buschbom-Helmke, Silke; Fox, Thomas; Gargano, Emanuele M.; Grabowski, Daniel; Hoenke, Christoph; Matera, Damian; Mueck, Katja; Peters, Stefan; Reindl, Sophia; Riether, Doris (2023-02-23). "Discovery of a Novel Potent and Selective HSD17B13 Inhibitor, BI-3231, a Well-Characterized Chemical Probe Available for Open Science". Journal of Medicinal Chemistry. 66 (4): 2832–2850. doi:10.1021/acs.jmedchem.2c01884. ISSN 0022-2623. PMC 9969402. PMID 36727857.
Further reading
[edit]- de Lange O, Wolf C, Thiel P, Krüger J, Kleusch C, Kohlbacher O, Lahaye T (November 2015). "DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats". Nucleic Acids Research. 43 (20): 10065–80. doi:10.1093/nar/gkv1053. PMC 4787788. PMID 26481363.
- Linke P, Amaning K, Maschberger M, Vallee F, Steier V, Baaske P, et al. (April 2016). "An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery". Journal of Biomolecular Screening. 21 (4): 414–21. doi:10.1177/1087057115618347. PMC 4800460. PMID 26637553.