In addition to the problem with high ionic strength pointed out by Evgeny Fadeev, there are several are problems that need to be considered when obtaining a spectrum of a protein in 3M Guanidinium hydrochloride:

1) High viscosity: solutions of 3M Guanidinium hydrochloride have a very high viscosity - this implies that the signals of the protein will be much broader than in the case of the same protein in solutions containing salts such as sodium chloride. Best way to deal with this problem is to increase the temperature of the experiment and if possible decrease the Gu.HCl concentration. Increasing the temperature will help to maintain the protein in the unfolded state.

2) High dynamic range: the protein concentration is 250 uM which is ~10000 times less than the concentration of the Guanidinium hydrochloride. Therefore, in addition to solvent suppression (suppression of the water signal), it would be necessary to suppress the hugh signal from Gu.HCl. There are several possible solutions to this problem: diffusion editing, editing based on spin-system type, chemical shift selective pulse, etc.

Diffusion editing: The molecular mass of Gu.HCl is much smaller than that of the protein, therefore, its translational diffusion coefficient should be higher. Appending a diffusion editing sequence in front of the HSQC will reduce the intensity of the Gu.HCl peak. However, Gu.HCl has an extensive network of hydrogen bonds, and as a result its translational diffusion coefficient is much lower than that of most other molecules of comparable (formal) size.

Shaped pulse: The last 180 composite proton pulse in the watergate HSQC may be modified such that inversion occurs for (almost) all protons except those of water and Gu.HCl.

Spectral editing/Spectral filter: Formally, the three nitrogen atoms in Gu.HCl have two attached protons each. There are a number of 1H-15N correlation experiments designed to select for the amide-NH and to suppress -NH2 signals (these were intended to suppress the signals from the side chain amides of asparagine and glutamine). Therefore, Use of these 1H-15N correlation experiments may reduce the intensity of the Gu.HCl signals. (You may also want to look at the standard DEPT and INEPT editing pulse sequences used in 13C NMR for selecting signals from heteronuclei attached to single proton while suppressing signals form heteronuclei attached to more than one proton. These pulse sequences could be easily modified for selective 1H-15N correlation.).

3) Chemical exchange: The hydrogens attached to the nitrogens of Gu.HCl exchange with the solvent hydrogens, and the rate of exchange is very high. This will give rise to complications for pulse sequences based on selective excitation as well as for the spectral editing pulse sequences, mentioned above.

BTW, I do not think that the protein signals are antiphase? (out of phase?) - it is your Gu.HCl signal that is out-of-phase - the spectrum should be phased such that the protein signals are in-phase.

BTW2, if you are only interested in measuring the rate at which folded proteins undergo denaturation, it would not be necessary to use NMR spectroscopy. You could accomplish the same by using UV absorbance spectroscopy. The kinetics of unfolding of proteins due to addition of Gu.HCl have been studied extensively using stopped flow kinetic experiments by the groups of Scheraga and Baldwin. The time resolution of these experiments (milliseconds) may be an advantage. If you are trying to identify protein folding intermediates, it would be helpful to carry out some preliminary stopped flow kinetic experiments monitored by UV/fluorescence to determine the optimum conditions that maximize the concentration of the observable protein folding intermediates.

In addition to the problem with high ionic strength pointed out by Evgeny Fadeev, there are several are problems that need to be considered when obtaining a spectrum of a protein in 3M Guanidinium hydrochloride:

1) High viscosity: solutions of 3M Guanidinium hydrochloride have a very high viscosity - this implies that the signals of the protein will be much broader than in the case of the same protein in solutions containing salts such as sodium chloride. Best way to deal with this problem is to increase the temperature of the experiment and if possible decrease the Gu.HCl concentration. Increasing the temperature will help to maintain the protein in the unfolded state.

2) High dynamic range: the protein concentration is 250 uM which is ~10000 times less than the concentration of the Guanidinium hydrochloride. Therefore, in addition to solvent suppression (suppression of the water signal), it would be necessary to suppress the hugh signal from Gu.HCl. There are several possible solutions to this problem: diffusion editing, editing based on spin-system type, chemical shift selective pulse, etc.

Diffusion editing: The molecular mass of Gu.HCl is much smaller than that of the protein, therefore, its translational diffusion coefficient should be higher. Appending a diffusion editing sequence in front of the HSQC will reduce the intensity of the Gu.HCl peak. However, Gu.HCl has an extensive network of hydrogen bonds, and as a result its translational diffusion coefficient is much lower than that of most other molecules of comparable (formal) size.

Shaped pulse: The last 180 composite proton pulse in the watergate HSQC may be modified such that inversion occurs for (almost) all protons except those of water and Gu.HCl.

Spectral editing/Spectral filter: Formally, the three nitrogen atoms in Gu.HCl have two attached protons each. There are a number of 1H-15N correlation experiments designed to select for the amide-NH and to suppress -NH2 signals (these were intended to suppress the signals from the side chain amides of asparagine and glutamine). Therefore, Use of these 1H-15N correlation experiments may reduce the intensity of the Gu.HCl signals. (You may also want to look at the standard DEPT and INEPT editing pulse sequences used in 13C NMR for selecting signals from heteronuclei attached to single proton while suppressing signals form heteronuclei attached to more than one proton. These pulse sequences could be easily modified for selective 1H-15N correlation.).

3) Chemical exchange: The hydrogens attached to the nitrogens of Gu.HCl exchange with the solvent hydrogens, and the rate of exchange is very high. This will give rise to complications for pulse sequences based on selective excitation as well as for the spectral editing pulse sequences, mentioned above.

BTW, I do not think that the protein signals are antiphase? (out of phase?) - it is your Gu.HCl signal that is out-of-phase - the spectrum should be phased such that the protein signals are in-phase.

BTW2, if you are only interested in measuring the rate at which folded proteins undergo denaturation, it would not be necessary to use NMR spectroscopy. You could accomplish the same by using UV absorbance spectroscopy. The kinetics of unfolding of proteins due to addition of Gu.HCl have been studied extensively by the groups of Scheraga, Baldwin and others. The time resolution of the order of milliseconds can be achieved by using stopped flow kinetic experiments by the groups of Scheraga and Baldwin. The time resolution of these experiments (milliseconds) may be an advantage. experiments. If you are trying to identify protein folding intermediates, it would be helpful to carry out some preliminary stopped flow kinetic experiments monitored by UV/fluorescence to determine the optimum conditions that maximize the concentration of the observable protein folding intermediates.intermediates.

In addition, it is possible to monitor the folding process indirectly. Monitor the extent of H/D exchange in the refolded protein by NMR spectroscopy, after allowing the protein to refold in a non-Gu.HCl buffer, after exposing the protein to Gu.HCl in D2O for a fixed period of time. (See work by Roder and Wuthrich on BPTI for this concept).