期刊
BIOMOLECULES
卷 10, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/biom10101458
关键词
huntingtin protein; low-complexity regions; nuclear magnetic resonance (NMR); cell-free protein expression; isotopic labeling; site-specific isotopic labeling; nonsense suppression
资金
- European Research Council under the European Union [648030]
- Labex EpiGenMed, an Investissements d'avenir program [ANR-10-LABX-12-01]
- GPCteR [ANR-17-CE11-0022-01]
- French National Research Agency [ANR-10-INBS-04-01, ANR-10-INBS-05]
- European Research Council (ERC) [648030] Funding Source: European Research Council (ERC)
The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics.
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