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I have determined the structure of a 110 residues protein(11kDa) which is known to interact with a 15mer peptide. Now I am interested to know which residues of the 15mer peptide interacts with this 11kDa protein. Can anyone suggest a simple nmr experiment which can tell the residues from the 15mer peptide which interact with the protein.

asked Jan 31 '11 at 22:38

man05's gravatar image


Hello, maybe you could try to come up with a better title? Try to make it more specific. Thanks! - Evgeny Fadeev (Jan 31 '11 at 23:01)

6 Answers:
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The answer certainly depends on the association and dissociation (kon/koff) rates as Paul Driscoll has suggested. In addition, the availability of isotope labeled samples for protein and/or peptide will be a major factor in deciding the type of experiments.

However, the following experiments would be expected to yield usable results over a wide range of kon/koff rates.

1) Protein titration: Carry out a series of experiments (TOCSY or HSQC or NOESY) keeping the ligand concentration fixed, with variable protein concentration.

You would expect that a new peak with intensity proportional to the total concentration of the complex would appear for resonance signals of residues that bind tightly (slow exchange).

You would expect that the chemical shift changes as the ligand:protein concentration changes for resonance signals of residues that bind weakly (fast exchange).

There will be a line broadening and the signal might even be wiped out for residues that are in intermediate exchange.

You may observe that different nuclei of the same residue are in different exchange rate regimes, i.e., some resonances of a residue may indicate slow exchange whereas other resonances of the same residue may indicate intermediate/fast exchange. This is because the designation of slow/intermediate/fast exchange depends on both the rate of exchange as well as the difference in chemical shifts of the free ligand and the bound complex. To determine the relative binding affinities of different aminoacids of the same peptide, it might be best to rely on results obtained by comparing the data for the backbone amide NH protons.

The major problem with this experiment would be that it is, in general, difficult to prepare samples with a wide variation in protein concentrations. It is usual to do the complimentary experiment, i.e., keep the protein concentration fixed and vary the ligand concentration (ligand titration e.g. Peili Zhang, Sekhar Talluri, Haiyan Deng, Daniel Branton and Gerhard Wagner. Structure, Volume 3, Issue 11, 1185-1195, 1 November 1995).

Experiments of this type assume that binding leads to a change in chemical shift - although it is true in most cases, it is not necessary.

2) The following experiment is based on the fact that the rate of H/D exchange is expected to be slower in a complex than in the free ligand. Obtain a 2D NMR spectrum (HSQC if 15N labeled sample is available, TOCSY/NOESY otherwise) on a sample containing 50ul D2O and 400ul H2O. Add 400ul of D20 and repeat the same experiment, immediately. Amide HN that are buried within the complex would be expected to exchange with solvent deuterons slowly, whereas amide HN of the peptide that are not involved in the complex would be expected to exchange with the solvent deuterons rapidly. Repeat the experiment at a series of intervals (preferably 1/2 hour intervals if your spectrometer is good enough). The intensities of the amide HN that are not involved in complex formation are expected to reduce more rapidly than those of HN that are involved in complex formation (final intensity will be ~1/4 of initial intensity). For a very stable complex, there may be no loss of signal intensity in the time period of your study (after the dilution factor has been corrected for).

This experiment exploits the competition between two dynamical processes: exchange (HN/DN) and dissociation of the complex. It is possible to tune the rate of H/D exchange over a wide range of values by proper choice of the pH. In addition the ratio of ligand:protein concentrations can be varied. As a consequence, this experiment has the ability to probe interactions over a very wide range of dissociation rates.

The primary limitation of this experiment is that only exchangeable hydrogen atoms of the peptide/protein can be used a probes of binding.

3) If isotope labeled samples are available, a wide variety of isotope editing and isotope filtering experiments will be possible, as others have mentioned before.

Other experiments based on the transferred NOE principle are also available, but these are limited to specific ranges of dissociation rates. (See Ref. Feng Ni, Harold A. Scheraga Acc. Chem. Res., 1994, 27 (9), pp 257–264).

Measurement of relaxation rates and dipolar coupling constants can also provide valuable information regarding the residues that are involved in binding.


answered Mar 02 '11 at 09:41

sekhar%20Talluri's gravatar image

sekhar Talluri

updated Mar 02 '11 at 10:19

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Depends on exchange rate:

If fast/intermediate exchange then look for differential line-broadening in peptide spectrum when peptide is in excess. If slow exchange then might need to isotope label the peptide to see which resonances are shifted and/or broadened. In this context it helps to know the binding affinity by some orthoganol method. With a 'receptor' of only 11 kDa it might be possible to filter the spectrum to see bound peptide chemical shifts (assuming the 11kDa molecule is isotope labelled).


answered Feb 02 '11 at 04:23

Paul%20Driscoll's gravatar image

Paul Driscoll

updated Feb 02 '11 at 04:23

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Differential line-broadening can be difficult to interpret in term of interaction topology. With a protein 15N labelled and a no labelled peptide, you can try a filtered-NOESY that give you distance between 14N-H of the peptide and 15N-H of the protein (other intramolecular NOE will be cancelled)...


answered Feb 22 '11 at 10:37

Yoan%20Monneau's gravatar image

Yoan Monneau

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If you know that peptide is interacting with your protein, you can simply run a nutural abundance 2D 15N-HSQC on your peptide. Then carry out a series of titrations by adding the unlabeled protein to the peptide. You can cleary see the movement of peptide peaks in the HSQC if there is an interaction.



answered Mar 04 '11 at 09:19

Ilango's gravatar image


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In most of the docking cases the saturation transfer techniques have been useful. But if the 15mer peptide peaks are all coinciding with some or the other of the protein with which interacts than, more attention is required. The saturation transfer (from the docked ligand to the protein host) mechanism is not the same mechnism as by which the chemical shifts coincide. Thus still this saturation trnasfer can be giving significant information.



answered Mar 06 '11 at 09:02

SankarampadiAravamudhan's gravatar image


updated Mar 06 '11 at 09:06

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Does anybody need some more services about protein structure to save your time, such as protein purification, crystallization, structure determination and function analysis with EM, NMR, etc.


answered Jun 27 '16 at 22:44

Justin%20Frank's gravatar image

Justin Frank

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