Differential Labelling Schemes and Isotope Filtering Nomenclature Labollod with N Unlabelled C,N 38eot8peeee tope-separated ←→ isotope-filtered 2D isotope-filtered(f1), 3D ope-edit Cdc42 ACK Free 15N-abelled ACK 15N-ACK+unlabelled Cdc42 1120 140 14. 16.0 118.0 120.0 1220 12 1240 124.0 126.0 126. 05b0508507店70ds 0.50050085&.051065
Differential Labelling Schemes and Isotope Filtering H H 13C H 13C 12C H 12C Labelled with 13C, 15N Unlabelled 12C, 14N 15N H 2D isotope-edited 3D isotope-separated isotope-filtered 2D isotope-filtered (f1), isotope-edited (f2) 3D isotope-filtered (f1), isotope-separated (f2/f3) H 14N Nomenclature Cdc42 + ACK Cdc42/ACK Complex Free 15N-labelled ACK 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 15N-ACK + unlabelled Cdc42 110.0 112.0 114.0 116.0 118.0 120.0 122.0 124.0 126.0 110.0 112.0 114.0 116.0 118.0 120.0 122.0 124.0 126.0
Protein Complexes:Slow Exchange (the ideal case) Chemical Shift Assignments CC(CO)NH,H(CCCO)NH,HBHA(CBCA)(CO)NH on each component of the complex 3.Coupl ncm thecom onstant experiments Tight Complexes:Slow Exchange (the non-ideal case) Chemical Shift Assignments 1.Backbone experiments require full deuteration and TROSY: N..HNCACB.HNCOICACB. 2.Sidechain experiments deuterated sample) SYarat NOESY(uhift of sidechainH) e not work.use all shifts (including CO)to put into Talos Full set of assignments of the free protein(s)is necessary
Chemical Shift Assignments 1. Backbone experiments: HSQC, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH recorded on each component of complex 2. Sidechain experiments: 15N-separated TOCSY, HCCH-TOCSY CC(CO)NH , H(CCCO)NH, HBHA(CBCA)(CO)NH on each component of the complex 3. Coupling constant experiments: HNHA on each component of the complex Protein Complexes: Slow Exchange (the ideal case) Chemical Shift Assignments 1. Backbone experiments require full deuteration and TROSY: HSQC, HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, HNCO, HNCACO 2. Sidechain experiments: CC(CO)NH (on deuterated sample) On protonated sample: HCCH-TOCSY, 15N-separated NOESY (to guess shifts of sidechain 1H) 3. Coupling constants: experiments do not work, use all shifts (including CO) to put into Talos. Tight Complexes: Slow Exchange (the non-ideal case) Full set of assignments of the free protein(s) is necessary