Hello, I would like to acquire an HSQC spectrum of an 15N-labeled 12 kDa protein unfolding (over the course of hours) in 3M GuaHCl. I have access to a Varian 600 spectrometer with a conventional probe. Due to the high ionic strength of the sample, tuning and shimming is a little difficult. However, I can record a sort-of-decent HSQC using a WaterGate gradient enhanced WGgNhsqc sequence. I am limited to using a protein concentration of about 250 uM. I've determined the pw90 to be 12.125 at a tpwr of 63. Using WGgNhsqc, the protein signal is decent (relative to the guanidine), but there is high phase distortion of the guanidine signal at 6.95 ppm. The guanidine signal is "split" half up and half down. I get slightly different results using a regular gNhsqc -- the protein seems suppressed relative to the guanidine signal, and "antiphase" to it. I am looking for ways of increasing sensitivity -- I don't mind if the HSQC takes an hour. Does anyone have any practical advise for such an experiment? Is one pulse sequence going to be significantly superior to another in this case? asked Oct 22 '10 at 15:04 vadim |
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 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. 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, 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). answered Oct 28 '10 at 12:36 sekhar Talluri |
To add to Evgeny's comment about small diameter tubes, if the salt concentration is very high, the losses in going to smaller diameter tubes (while keeping the concentration constant) will likely be somewhat smaller than you you would expect from the change in sample amount. A 3mm tube would use about 1/3 the volume of a 5mm tube, but if the pulse length in the 3mm tube is only 2/3 of the pulse length in the 5mm tube at the same power level then you get a factor of 3/2 increase in signal in the 3mm tube (principle of reciprocity). Thus, overall you would only lose half your signal rather than two thirds. In addition, the sample noise will be reduced, so overall s:n loss will be even less. Furthermore, you have a shorter 90 degree pulse, which is generally beneficial. These things are normally more an issue with cryoprobes, but due to the very high salt concentrations involved this may be worth looking at even in a room temperature probe. I have never worked with such high salt concentrations so can't be sure how things would compare between 3 and 5mm tubes in your case, but you should certainly try. What is the 90 degree pulse for a sample with no salt? Also, if I read your comment correctly, you have measured this pulse at full power (tpwr=63). I guess that your 90 degree pulse without salt at this power would be very short - I seem to recall seeing numbers like 7us at tpwr=58 for similar setups. Using full power may not be a good idea - I don't know anything about the specification of Varian probes, but this is something you should be careful of. answered Oct 30 '10 at 06:29 Pete Gierth |
The problem with high-salt (high electric conductivity) samples is heating losses due to the stronger coupling of sample with the coils. As more RF energy is absorbed by the ions in the sample, less becomes available to measure the NMR signal, so signal to noise ratio falls. One way to reduce that coupling is to use smaller volume samples by using smaller diameter tubes. For example, this paper shows that NMR can be done in 3.5 M NaCl. Another method is to use low conductivity buffer (e.g. HEPES) to keep ionic strength high, while reducing the losses to conductivity, but if you have to use guanidine, this method is probably not be an option for you. Maybe you can benefit from shimming on lineshape of some small molecule - like 3-(trimethylsilyl)propane sulfonic acid (TSP), with better shims water suppression will improve. High salt conditions are frequently used for NMR of nucleic acids, you'll probably find more information in the papers on NMR studies of DNA/RNA. Looks like with some extra measures you can use standard pulse sequences for the high salt work. answered Oct 27 '10 at 15:29 Evgeny Fadeev |
from what I get in the answers tagged "salt" sequences work better for salty samples when hard pulses are used for the water suppression and shims are extensively optimized. Hopefully someone will offer more informative answer... - Evgeny Fadeev (Oct 22 '10 at 15:54)
if guanidine signal is antiphase, maybe there is a way to cancel it with some phase cycle? - Evgeny Fadeev (Oct 27 '10 at 15:53)