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Friday, May 8, 2015


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.
At very high fields, One of the lines in the 1H-15N doublet is very sharp and the other very broad.  If, in the 1H spectrum of a protein, we could eliminate all of the broad doublet components leaving only the sharp ones, we would have a high resolution 1H spectrum.  Further, if we could combine such a measurement with an 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.
Clearly, it is not advantageous to use the TROSY technique on small proteins rather than the conventional 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.
One can clearly see the ½ 1JHN shift in the F2 and F1 domains of the TROSY compared to the 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

Decoupling in 2D HSQC Spectra

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.
During the evolution time, t1, the 13C chemical shift and 1H-13C coupling evolve.  The 1H 180° pulse (color coded in yellow) in the center of the evolution time refocuses the coupling and as a result decouples protons in the F1 (13C) domain of the spectrum.  13C is broadband decoupled from the F2 (1H) domain by applying a GARP pulse train (color coded in pink) at the 13C frequency during the collection of the FID.  One can turn each of these elements “on” or “off” for data collection.  The figure below shows the 1H-13C gradient HSQC spectrum of benzene with all possible combinations of 1H and/or 13C decoupling.
In the top left panel both 1H and 13C decoupling are turned “on” and one observes a singlet in both the F2 (1H) and F1 (13C) domains.  In the top right panel, the 1H decoupling element is “on” while the 13C decoupling element is “off”.  The result is a 1H-13C doublet in the F2 (1H) domain and a singlet in the F1 (13C) domain.  In the bottom left panel, the 1H decoupling element is “off” while the 13C decoupling element is “on”.  The result is a 13C-1H doublet in the F1 (13C) domain and a singlet in the F2 (1H) domain.  In the bottom right panel, both the 1H and 13C decoupling elements are “off”.  The result is a 1H-13C doublet in both the F2 (1H) and F1 (13C) domains.