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Advanced J-Couplings Assignment for 13C/15N Labelled Proteins in Heteronuclear Experiments

This assignment explores advanced J-coupling techniques in heteronuclear experiments involving 13C and 15N labelled proteins. It delves into the intricacies of 1H, 15N, and 13C decoupling methods, and highlights two significant issues: 13C Jcc coupling and spectral width constraints. The employment of constant-time HSQC and targeted pulse techniques to optimize spectral resolution and transfer efficiency is examined. This comprehensive analysis aims to enhance the understanding of J-coupling interactions and their impact on protein assignment strategies.

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Advanced J-Couplings Assignment for 13C/15N Labelled Proteins in Heteronuclear Experiments

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  1. Assignment II

  2. Assignment using J-couplings 13C/15N labelled proteins

  3. Heteronuclear Experiment D/2 x y y 1H t2 f y t1 15N Decoupling f=x,-x y=x,x, -x, -x frec= x, –x, –x, x D=1/2JNH

  4. 13C-HSQC D/2 x y y 1H t2 f y t1 13C Decoupling D=1/2JCH Two new issues: 1) 13C Jcc coupling (~35Hz): i.e. for long t1 (~1/Jcc=28ms) CC coupling resolved 2) 13C spectral width is ~160ppm (~25Khz at 600 MHz)

  5. Constant time HSQC 2T=n/Jcc D/2 x y y 1H t2 f y T-t1/2 T t1/2 13Caliph Decoupling 13CO 15N

  6. Excitation profile of a pulse The frequency response of a pulse is the Fourier transform of the pulse tp w t Dw ~ 1/tp 10us pulse => Dw~105Hz i.e. 10x the spectral with of a eg protons at 600 HMZ in effect all protons experience the same pulse (flip angle) Vice versa: Long pulses are frequency selective: 1s pulse => Dw~1Hz i.e. very frequency selective (e.g. used in selective water suppression)

  7. Spectral width of 13C and shaped pulses 13C spectral width is ~160ppm (~25Khz at 600 MHz) Hence difficult to obtain homogenous excitation (or inversion) of all nuclei using rectangular pulses

  8. HNCO/HNCA Experiment D/2 f1 y y D D 1H t3 f2 f3 y t2/2 t t-t2/2 t t 15N Decoupling f4 f5 t1 13Ca/CO 13Ca/CO f1=y, f2=x, F3=2(x) 2(-x), F4=x+BSP, F5=x,-x + TPPI; frec= x, 2 (–x),2 (–x), x D=1/2JNH (5.4ms) ; t=1/2JNCA (13.5ms) or t=1/2JCO (11ms)

  9. Assignment Experiments

  10. Example

  11. Cb Cb Ca N CO Ca H H H H H H H HNCACB CACB(CO)NH Residue i Residue i-1 Assignment strategy for 13C/15N labelled proteins: Triple resonance (1H-15N-13C)

  12. NOE, J-couplings 2nd Ca shifts

  13. Transfer efficiency using J couplings

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