“CON-CON” assignment strategy for highly flexible intrinsically disordered proteins
- 556 Downloads
Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel 1H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on 13C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.
KeywordsIntrinsically disordered proteins 13C detection 1HN detection 1Hα detection NUS Multidimensional NMR experiment BEST-TROSY Backbone assignment
This work has been supported in part by the European Commission Projects IDPbyNMR (Contract No. 264257), BioNMR (Contract No. 261863) and INSTRUCT (Contract No. 211252).
- Felli IC, Piai A, Pierattelli R (2013) Recent advances in solution NMR studies: 13C direct detection for biomolecular NMR applications. Ann Rep NMR Spectroscop 80:359–418Google Scholar
- Goddard TD, Kneller DG (2000) SPARKY 3. University of California, San FranciscoGoogle Scholar
- Motáčkova V, Nováček J, Zawadzka-Kazimierczuk A, Kazimierczuk K, Židek L, Sanderová H, Krásný L, Koźmiński W, Sklenář V (2010) Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments. J Biomol NMR 48:169–177CrossRefGoogle Scholar
- Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T-2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar