Abstract An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely 13Câ?²(i), 15N(i), 1HN(i) connectivities in uniformly 15N/13C-labeled proteins. In comparison to the â??out-and-backâ?? style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the 13Câ?² dimension and on average 1.6 times higher sensitivity especially for residues in α-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16.
Content Type Journal Article
Pages 301-310
DOI 10.1007/s10858-009-9373-4
Authors
Sampo Mäntylahti, University of Helsinki Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory P.O. Box 65 00014 Helsinki Finland
Helena Tossavainen, University of Helsinki Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory P.O. Box 65 00014 Helsinki Finland
Maarit Hellman, University of Helsinki Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory P.O. Box 65 00014 Helsinki Finland
Perttu Permi, University of Helsinki Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory P.O. Box 65 00014 Helsinki Finland
Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins
Extension of the HA-detection based approach: (HCA)CON(CA)H and (HCA)NCO(CA)H experiments for the main-chain assignment of intrinsically disordered proteins
Abstract Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented by utilizing 15N, 13C� and 1HN spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues. Especially 15N and 13C� spins offer superior chemical shift dispersion in comparison to 13Cα and 13Cβ spins. However, HN-detected experiments...
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Abstract Traditional single site replacement mutations (in this case, phenylalanine to tyrosine) were compared with methods which exclusively employ 15N and 19F-edited two- and three-dimensional NMR experiments for purposes of assigning 19F NMR resonances from calmodulin (CaM), biosynthetically labeled with 3-fluorophenylalanine (3-FPhe). The global substitution of 3-FPhe for native phenylalanine was tolerated in CaM as evidenced by a comparison of 1H-15N HSQC spectra and calcium binding assays in the presence and absence of 3-FPhe. The 19F NMR spectrum reveals six resolved resonances, one...
An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins
Linear Mode
