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### Topic: Question about selective saturation (STD and 1D-NMR)  (Read 1336 times)

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#### riboswitch

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##### Question about selective saturation (STD and 1D-NMR)
« on: November 20, 2018, 10:09:13 AM »
I am having a bit of a problem understanding what "selective saturation" is supposed to mean when it comes to the one-dimensional NMR technique which is called "Saturation Transfer Difference" or STD. I only understood the concept of saturation when I was studying what is the process of spin relaxation in NMR. If I remember correctly, saturation is a phenomenon that occurs when the Boltzmann equilibrium distribution of nuclear spins are perturbed such that the population of the α spins (Nα) are equal to the population of the β spins (Nβ). However, I have no idea whether this is the same "saturation" concept used in Saturation Transfer Difference, used to detect transient protein-ligand binding.

What little I understand about STD is the following: on-resonance spectrum and off-resonance spectrum of a sample are collected and a difference between them is determined. Subtraction between the two spectra will contain the resonances of the small molecule when there is interaction between the ligand and the protein. But I just don't understand how the phenomenon of saturation plays a role in all of this.

Thanks in advance for the help.

#### Corribus

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##### Re: Question about selective saturation (STD and 1D-NMR)
« Reply #1 on: November 20, 2018, 11:00:16 AM »
I am by no means an NMR theory specialist. With that in mind, from my understanding, the proton signals in the protein are selectively saturated (based on the definition you provided). Spin diffusion results in desaturation, if you will, of the protein signals faster than intrinsic relaxation timescale and simultaneous activation of nearby coupled proton spins. This process depends on the distance of the ligands from the activated protein protons. A somewhat crude analogy would be FRET in fluorescence spectroscopy, if you are familiar with that.

Have you seen this paper published in J. Chem. Educ.?
https://pubs.acs.org/doi/10.1021/ed101169t
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

#### riboswitch

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##### Re: Question about selective saturation (STD and 1D-NMR)
« Reply #2 on: November 20, 2018, 12:05:01 PM »
Have you seen this paper published in J. Chem. Educ.?
https://pubs.acs.org/doi/10.1021/ed101169t

Thank you! So, quick definitions from that paper:
• Off-resonance spectrum - This refers to the spectrum that is recorded without selective saturation of the protein protons. The signal intensities of this spectrum is referred by the paper as I0;
• On-resonance spectrum - This refers to the spectrum that is recorded with selective saturation of the protein protons. The signal intensities of this spectrum is referred by the paper as ISAT;
From the two spectra, a difference spectrum is determined through subtraction between the signal intensities of the off-resonance spectrum and on-resonance spectrum:
$$I_{STD} = I_{0}-I_{SAT}$$
In the difference spectrum, only the signals of the ligand that received saturation transfer from the protein will remain. According to the paper, the saturation transfer from protein to ligand occurs via spin diffusion, through the so-called nuclear Overhauser effect. If I remember correctly, NOE is the modification of the signal intensity of a resonance by saturation of another. For example, in a very simple AX system in which the two spins interact through a magnetic dipole-dipole interaction, if we saturate the transition of X (that is, we equalize the populations of the X levels), we observe that the signal intensity of A is either enhanced or diminished.

Also, according to the paper, in order to observe STD effects, the dissociation constant or KD of the protein-ligand complex must be lower than 10-3 M but higher than 10-8 M. If the ligand binds more tightly than the acceptable range of the KD required for the experiment to work, then according to the paper, relaxation occurs and saturation transfer does not occur.

Have I understood and summarized the concepts well?

Also, right now I'm confused about why saturation transfer doesn't occur when relaxation occurs. Why? (Sorry for asking stupid questions...)

#### Corribus

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##### Re: Question about selective saturation (STD and 1D-NMR)
« Reply #3 on: November 21, 2018, 12:08:02 PM »
Actually, I cannot access that paper. By the title it just sounded useful. Your summary sounds reasonable to me, though.

Regarding your final question - basically in NMR you are creating a nonequilibrium situation when you hit the sample with the Rf pulse. The protons then relax back down to the equilibrium state (reference state imposed by the static background magnetic field) with a characteristic amount of time once the Rf pulse is over. This relaxation, and the associated energy difference between the magnetic spin states, is what creates the NMR spectrum. In the STD experiment, the rate of relaxation competes with the rate of spin transfer to nearby nuclei in the ligands: if the transfer process is slower than the characteristic relaxation time, because - say - the ligands are too far away, then you won't observe any transfer. On the other hand, if the transfer process is faster than relaxation, then you will observe changes in the nuclear spins of the ligands. You basically have two process that lead to two end-points, and which process is faster determines what you observe in the experiment.

A crude analogy might be this: suppose you get a paycheck of 100 dollars, which creates a nonequilibrium state. The drive back to equilibrium (not having spending money) happens with a characteristic rate (average time it takes you to find something on Amazon to buy). There is a chance you may deposit the money in the bank, and the chance depends on how far away the bank is. You could express this also as an average rate of transfer of funds to the bank. If the bank is close, you are more likely to drive to the bank and deposit the money before you spend it somewhere (rate of transfer exceeds the rate of spending), and you will observe money appearing in your bank account. If the bank is far, the average amount of time it takes to get to the bank and deposit money exceeds the amount of time it takes to spend the money on junk (rate of spending exceeds the rate of transfer), and you rarely see a transfer of money to your bank. It's all about what rate dominates the overall process to determine what is the outcome you observe.
« Last Edit: November 21, 2018, 12:40:56 PM by Corribus »
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman