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.
Extensive resonance overlap exacerbates assignment of intrinsically disordered proteins (IDPs). This issue can be circumvented by utilizing (15)N, (13)C' and (1)H(N) spins, where the chemical shift dispersion is mainly dictated by the characteristics of consecutive amino acid residues. Especially (15)N and (13)C' spins offer superior chemical shift dispersion in comparison to (13)C(α) and (13)C(β) spins. However, HN-detected experiments suffer from exchange broadening of amide proton signals on IDPs especially under alkali conditions. To that end, we propose here two novel HA-detected experiments, (HCA)CON(CA)H and (HCA)NCO(CA)H and a new assignment protocol based on panoply of unidirectional HA-detected experiments that enable robust backbone assignment of IDPs also at high pH. The new approach was tested at pH 6.5 and pH 8.5 on cancer/testis antigen CT16, a 110-residue IDP, and virtually complete backbone assignment of CT16 was obtained by employing the novel HA-detected experiments together with the previously introduced iH(CA)NCO scheme. Remarkably, also those 10 N-terminal residues that remained unassigned in our earlier HN-detection based assignment approach even at pH 6.5 were now readily assigned. Moreover, theoretical calculations and experimental results suggest that overall sensitivity of the new experiments is also applicable to small or medium sized globular proteins that require alkaline conditions.
Program in Structural Biology and Biophysics, NMR Laboratory, Institute of Biotechnology, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland.
Sampo
S
Mäntylahti
Maarit
M
Hellman
Perttu
P
Permi
D002247:Carbon Isotopes / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins
J Biomol NMR. 2001 Jun;20(2):135-47
11495245
Protein Sci. 1997 Jun;6(6):1248-63
9194185
FEBS Lett. 1997 Dec 15;419(2-3):285-9
9428652
J Biomol NMR. 2005 Sep;33(1):43-50
16222556
J Biomol NMR. 2006 May;35(1):27-37
16791738
J Biomol NMR. 2010 Aug;47(4):243-8
20574814
Biomol NMR Assign. 2007 Jul;1(1):85-7
19636834
J Am Chem Soc. 2007 May 2;129(17):5484-91
17417840
J Biomol NMR. 2010 Jul;47(3):171-81
20437194
J Biomol NMR. 1994 Mar;4(2):301-6
8019138
J Magn Reson. 2011 Dec;213(2):423-41
22152361
J Biomol NMR. 1997 Jan;9(1):94-100
9081546
J Biomol NMR. 1993 Jan;3(1):113-20
8448431
J Biomol NMR. 2009 Nov;45(3):301-10
19768387
J Magn Reson. 2004 Oct;170(2):244-51
15388087
Nat Rev Mol Cell Biol. 2005 Mar;6(3):197-208
15738986
J Magn Reson. 2009 Jun;198(2):275-81
19307141
J Biomol NMR. 2002 Jul;23(3):201-9
12238592
Int J Cancer. 2011 May 15;128(10):2382-92
20658534
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12366-71
9356455
J Biomol NMR. 1999 Apr;13(4):381-5
10353197
Biophys J. 2010 May 19;98(10):2383-90
20483348
Bioinformatics. 2005 Aug 15;21(16):3433-4
15955779
Proteins. 1993 Sep;17(1):75-86
8234246
J Magn Reson B. 1996 May;111(2):194-8
8661281
J Biomol NMR. 1991 Nov;1(4):439-46
1841710
J Biomol NMR. 2000 Mar;16(3):253-9
10805132
Methods Enzymol. 2001;339:258-70
11462815