The chemical shift resolution and sensitivity of NMR generally benefit from an increase in magnetic field strength. As a result, large sums of money are spent on magnets with higher and higher fields. The boost in sensitivity means that smaller and smaller quantities of sample are needed and measurements can be completed in shorter periods of time. There are particular cases however, where an increase in magnetic field can lead to a loss of sensitivity and resolution. This is the case for 15N decoupled 1H spectra of the 1H-15N spin pairs in very large 15N labelled proteins. The very long correlation times of the protein combined with the high resonance frequencies associated with high field strength lead to very short T2 relaxation times and therefore broader lines. The broad lines account for a significant loss in resolution and sensitivity. One might then wonder why protein structural chemists spend so much money on very high field magnets. What follows is one possible answer to this question.
The two main relaxation mechanisms for the 1H and 15N in proteins are dipolar coupling and chemical shielding anisotropy. These two mechanisms are also cross correlated with one another. The cross correlation term is of different sign for each of the two peaks in a 1H-15N doublet resulting in one of the peaks of the doublet having a shorter T2 (and broader line) than the other one. If 15N decoupling is applied, one sees a single resonance with a line width determined by the average of the two components of the doublet. This is illustrated in the figure below for a 1H-15N spin pair in a small and large molecule at high field . The same is true in the 15N-1H doublets in the 15N spectra of 15N-1H spin pairs.
HSQC, we would have a high resolution 1H-15N HSQC at high field. The combination of these two measurements is called transverse relaxation optimized spectroscopy (TROSY). TROSY data collection employs an HSQC measurement with neither 1H nor 15N decoupling elements (as described in a previous post) as well as other elements which suppress the broad lines of the doublets and retain the sharp lines. The results of this are illustrated in the figure below for small and large proteins at high field.
HSQC. For large proteins at high field however, there is a significant sensitivity and resolution advantage compared to a conventional HSQC. It should be noted that the TROSY cross peaks are shifted by ½ 1JHN in both the F2 and F1 domains. The figure below shows a superposition of a conventional HSQC (black) and a TROSY (blue) for a protein at 500 MHz.
HSQC. In this case, the conventional HSQC gives higher sensitivity than the TROSY.
Thank you to Adam Damry of Professor Roberto Chica’s research group at the University of Ottawa for providing the sample of 15N labelled protein.
Wednesday, May 6, 2015
HMQC and HSQC NMR data are commonly used to correlate the chemical shifts of protons and 13C (or 15N) across one chemical bond via the J coupling interaction. The data are 1H detected, with the 1H chemical shift in the horizontal F2 domain and the 13C (or 15N) chemical shift in the vertical F1 domain. In the case of 1H and 13C, the technique depends on protons bonded to 13C. 1H–12C spin pairs provide no coupling information and are suppressed by the method. If one is to observe the 1H signal of a 1H-13C spin pair, one expects to observe a doublet with splitting 1JH-C (i.e. the 13C satellites). Likewise, if one is to observe the 13C signal of a 1H-13C spin pair, one expects to observe a doublet with the same splitting. 2D HSQC spectra are normally presented with both 1H and 13C decoupling yielding a simplified 1H-13C chemical shift correlation map over one chemical bond. The figure below shows one of the most commonly used gradient HSQC pulse sequences. The 1H and 13C decoupling elements of the sequence are highlighted in yellow and pink, respectively.