3D HNHB

The main use of this experiment is to correlate the amide proton (HN) and nitrogen resonances with side chain HB proton signals via the quantitative J-correlation method based on the quantification of the in-phase and anti-phase components resulting from evolution of J coupling. This method will enable the user to rapidly and accurately measure the ³J(NH^β) and ³J(NH^α) as compared to the older 2D version which requires a separate 2D reference experiment to be recorded.

The ³J(NH^β) and ³J(NH^α) information will give information regarding the side chain angle (Fig. 1) and backbone angle (Fig. 2) respectively and thus allow the user to better validate large and complicated protein.

Side Chain angle:

Backbone Angle:

3D HNHB Pulse Scheme

3 different sources of the pulse scheme will be mentioned in this report while only the original pulse scheme (Fig. 3) from the original source will be elaborated. The other 2 pulse scheme are from the blue book (Fig. 4) and bruker database (Fig. 5).

In Fig.below (A) shows the regular pulse scheme, t₁ evolution period precedes the ¹⁵N-Hβ hetero-nuclear multiple-quantum correlation (HMQC) fragment (between vertical dashed lines) while (B) shows a constant-time version of this HNHB experiment, where the t₁ evolution period has been integrated in the first Δ delay of the ¹⁵N-Hβ HMQC. Narrow and wide pulses represent 90” and 180” flip angles. Unless marked, all pulses are applied along the x axis.

Original HNHB pulse Scheme:

Bruker Pulse program:

Advantage over other methods

Although values of ¹⁵N-¹H spin-spin coupling can be measured quantitatively by NOSEY spectra of a ¹⁵N enriched protein in the absence of of ¹⁵N decoupling or ¹H-¹H TOCSY/HOHAHA . However, the lack of ¹⁵N decoupling halves the signal to noise ratio and may make the small displacement of broad resonance which contains the long range J(NH) information difficult to measure when using NOESY. Also, for larger protein, TOCSY/HOHAHA cross peak are usually too weak to be detected at all. (NOE effect larger for large molecules and almost similar to J coupling)

References

Archer, S. J., Ikura, M., Torchia, D. A., & Bax, A. (1991). An alternative 3D NMRtechnique for correlating backbone 15 N with side chain Hβ resonances in largerproteins. Journal of Magnetic Resonance (1969), 95(3), 636-641.

Düx, P., Whitehead, B., Boelens, R., Kaptein, R., & Vuister, G. W. (1997). Measurement of 15N-1H coupling constants inuniformly 15N-labeled proteins: Application to the photoactive yellow protein. Journal of biomolecular NMR,10(3), 301-306.

Barnwal, R. P., Rout, A. K., Chary, K. V., & Atreya, H. S. (2007). Rapid measurement of3J (HN–Hα) and 3J (N–Hβ) coupling constants in polypeptides. Journal of biomolecular NMR, 39(4), 259-263.