Sample Preparation And Water Suppression

Water is the most relevant biological solvent. The amide protons (one for each peptide linkage -HN-CO-) exchange with solvent water so it is not desirable to use D2O because the HN becomes DN and can no longer participate in NMR experiments. Usually the solvent is a mixture of 90% H2O and 10% D2O, with the D2O used for locking and shimming in the NMR magnet. This requires some very fancy methods of water signal suppression to remove the enormous H2O peak at 4.8 ppm (about 100 M in H2O protons vs. about 1 mM in protein).

12.4.1 Buffers and pH

Although the most relevant pH for most biological molecules is near 7, the exchange of amide protons ("HN") with solvent H2O is very rapid at neutral pH, leading to exchange broadening and weak or nonexistent NMR peaks for these protons. If we have fast enough exchange, the HN protons spend the vast majority of their time in the much larger pool of H2O protons and the average chemical shift is identical to the water chemical shift. The minimum exchange rate occurs at pH 2-3, and early protein NMR studies were done at this pH, but most of the work is now done between pH 4.5 and pH 7. Even at pH 7.5 life is getting difficult and 8.0 is a real challenge. Buffers for NMR include deuterated acetate (CD3CO2Na + HCl titrated to pH 4.5), sodium phosphate (NaH2PO4/Na2HPO4), and deuterated tris ((HOCD2)3CNH2 + HCl), typically at concentrations around 50 mM. Salt (NaCl) sometimes has to be added up to 100 mM in order to provide the proper ionic strength to solubilize the protein or prevent it from aggregating. Excessive salt is to be avoided if at all possible because it leads to poor matching of the probe circuit and lengthens the 90° pulse. With cryogenic probes salt is death to the sensitivity advantage you paid so much for, and special care should be taken to use the best buffer for cryogenic probe work. Usually sodium azide (NaN3, 1 mM) is added to prevent bacterial growth in the sample. Samples are stored in a refrigerator (5°C) and not frozen because freezing can break the NMR tube and denature the protein. Special tubes (Shigemi tubes) are often used to reduce the sample volume from 0.5 mL to around 0.3 mL (300 ^L) just within the probe coil, filling the remaining volume above and below the sample with a special glass whose magnetic susceptibility is matched to that of water.

12.4.2 Referencing

TMS cannot be used in aqueous solution because it is not water soluble. For a chemical-shift reference, a water-soluble equivalent (such as sodium d4-3-trimethylsilylpropanoate ((CH3)3SiCD2CD2CO2-Na+, "TSP")) can be added; the single 1H peak is defined as zero ppm in water. The water peak itself can also be used as a chemical-shift reference, but care must be taken to correct for the temperature dependence of its chemical shift. Referencing of 13C and 15N chemical shifts can be done by using an accurate 1H reference. If the exact chemical shift is known at the center of the 1H spectral window (usually the water resonance), the precise radio frequency can be calculated for the zero point of the 1H chemical-shift (ppm) scale. For example, on a 600 MHz spectrometer with a reference frequency of 600.13231564 MHz and a water chemical shift of 4.755 ppm:

= 600.13231564 MHz - 2853.62 Hz = 600.12946202 MHz

Using the exact values for the magnetogyric ratios of 1H, 13C, and 15N, we can calculate the frequencies corresponding to 0 ppm 13C and 0 ppm 15N:

v(8 = 0, 13C) = 600.12946202 MHz x 0.25144953 (yC/yH)

v(S = 0,15N) = 600.12946202 MHz x 0.101329118 (yN/yH)

It is important to use the most accurate values available for these ratios and to do the calculation on a computer spreadsheet to avoid truncation errors. Once you have the frequency corresponding to 0 ppm, the chemical shift at the center of the spectral window ("the carrier") can be calculated from the reference frequency of the 13 C or 15N channel as we did above for the 1H channel.

12.4.3 Radiation Damping

The water signal from a 90% H2O sample is unlike any other NMR peak in that it is incredibly intense, so intense that the FID signal in the probe coil is strong enough to turn around and act on the sample as a pulse! This "pulse" then rotates the net magnetization vector of water back toward the +z axis, effectively accelerating its transverse relaxation and broadening the water peak. The worst thing you can do to water is to put it on the -z axis: after a 180° pulse the water net magnetization is very close to -z, but never exactly on it. It begins to precess around the -z axis, and the tiny component in the x-y plane induces a strong FID in the probe coil, which in turn starts to rotate the net magnetization away from the -z axis (Fig. 12.6). As it rotates away, the component in the x-y plane increases, the FID signal increases, and the rate of rotation increases as a result. The process accelerates until the water magnetization reaches the x-y plane, where the FID signal in the probe is at a maximum and the rotation rate is the greatest. It continues precessing and rotating until it reaches the +z axis. If we view it in the rotating frame of reference with the water peak on-resonance, we see only the rotation part and no precession: the vector starts near the -z axis and rotates around the x! axis at a rate that accelerates until it reaches the y' axis and then slows down as it approaches the +z axis. Keep in mind that relaxation by the normal T1 and T2 processes cannot generate coherence: after a 180° pulse the z magnetization recovers from -Mo to +Mo in an exponential fashion, without any rotation of the net magnetization vector. Dilute solutions of water (e.g., in D2O) behave normally with quite long T1 and T2 times due to the small size of the H2O molecule (see Fig. 5.17), but pure water (or 90% water) relaxes very rapidly due to radiation damping, leading to a very broad water peak in the spectrum.

Radiation damping

Radiation damping

Off-resonance

On-resonance + z

Off-resonance

On-resonance + z t

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