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NMR Deliverables

Compound binding to small molecule/peptide (SBSM)

  1. 2D DQF-COSY - Double quantum filtered homonuclear correlation experiment used to identify which spins are directly coupled to one another. The DQF version of the COSY experiment is generally superior to the standard COSY due to the fact that the diagonal peak intensity is decreased, resulting in the cross peaks being less obscured.
  2. 2D TOCSY – with multiple spin lock times - Experiment used to identify spins connected by a chain of couplings (similar to the COSY experiment, but gives more correlations – this can be manipulated by changing the spin lock time)
  3. 2D NOESY – with multiple mixing times - This experiment relies on the Nuclear Overhauser cross relaxation between nuclear spins during the mixing period to establish spin correlations. The spectrum obtained is similar to COSY, with diagonal peaks and cross peaks, however the cross peaks connect resonances from nuclei that are spatially close rather than those that are through-bond coupled to each other.
  4. Natural abundance 15N HSQC - Heteronuclear single-quantum correlation spectroscopy (HSQC) HSQC detects correlations between nuclei of two different types, which are separated by one bond. This method gives one peak per pair of coupled nuclei, whose two coordinates are the chemical shifts of the two-coupled atoms. Typical HSQC experiments are collected on isotopically labeled. Samples (i.e. 15N or 13C). Natural abundance HSQCs requires longer data collection time as they rely on the weak signal arising from natural abundance 15N and 13C.
  5. Natural abundance 13C HSQC - See 1d

Compound binding to protein (SBPB)

  1. 15N HSQC (titration) - See 1d
  2. 15N-edited NOESY - 3D NOESY experiment (see 1c) providing spatial correlations between the amide proton and all other nearby protons.
  3. 15N-edited TOCSY - 3D TOCSY experiment (see 1b) providing “through-bond” connectivities within a single amino acid residue

NMR titration (SBNT)

  1. 15N HSQC series - See 1d
  2. If successful follow up with 2b and 2c

NMR chemical shift assignment – heteronuclear (SBNA_HT)

  1. 15N-edited NOESY - See 2b
  2. 15N-edited TOCSY - See 2c
  3. 3D HNCA - 3D experiment for sequence specific assignment of amide protons, amide nitrogen atoms and alpha carbons within a protein. The magnetization of the amide proton of an amino acid residue is transferred to the amide nitrogen, and then to the alpha carbons of both the starting residue and the previous residue in the protein's amino acid sequence. This chemical shift information can then be used together with that derived from other experiments to assign the entire protein.
  4. 3D HNCACB - 3D experiment similar to the HNCA (see 4c), but with magnetization transfer allowing for the determination of the beta carbon chemical shifts (along with the alpha carbon) for the current and preceding amide group.
  5. 3D CBCA(CO)NH - 3D experiment similar to the HNCA (see 4c), but with magnetization transfer allowing for the determination of the beta carbon chemical shifts (along with the alpha carbon) for the preceding amide group only.
  6. 3D H(CCO)NH - 3D experiment designed to correlate the 1H and 15N amide resonances of one residue with 1H and all other 1H side-chain resonances of its preceding residue via the intervening 13CO and 13C aliphatic spins. This is accomplished via an isotropic 13C mixing time that enables magnetization to be passed between the carbon nuclei.  This experiment is useful in assigning the proton chemical shifts of the amino acid side chains.
  7. 3D C(CO)NH - 3D experiment similar to the H (CCO)NH (4f) except instead of providing 1H chemical shifts, 13C chemical shifts are determined for the preceding amino acid residue.  This experiment is useful in assigning the remaining amino acid side chain carbons.
  8. 3D HNCO - Experiment in which magnetization is passed from 1H to 15N and then selectively to the carbonyl 13C via the 15NH-13CO J-coupling. Magnetization is then passed back via 15N to 1H for detection. The chemical shift is evolved on all three nuclei resulting in a three-dimensional spectrum. This is the most sensitive triple-resonance experiment. In addition to the backbone CO-N-HN correlations, Asn and Gln side-chain correlations are also visible.
    • It is mainly used to obtain CO chemical shifts that can be used in a program like TALOS to help predict secondary structure.
    • The HNCO can also be useful for backbone assignment in conjunction with the HN(CA)CO experiment.
  9. 3D HN(CA)CO - In this experiment the magnetization is transferred from 1H to 15N and then via the N-Cα J-coupling to the 13Cα. From there it is transferred to the 13CO via the 13Cα-13CO J-coupling. For detection the magnetization is transferred back the same way, resulting in a three-dimensional spectrum. Because the amide nitrogen is coupled both to the Cα of its own residue and that of the preceding residue, both these transfers occur and transfer to both 13CO nuclei occurs. Thus for each NH group, two carbonyl groups are observed in the spectrum. This experiment can be useful for backbone assignment when used in conjunction with the HNCA, HN(CO)CA and HNCO.

NMR chemical shift assignment – homonuclear (SBNA_HO)

  1. 2D NOESY – multiple mixing times - See 2c
  2. 2D TOCSY – multiple spin lock times - See 2b
  3. Natural abundance 15N HSQC - See 1d
  4. Natural abundance 13C HSQC - See 1d

NMR amide exchange – protein (SBNX)

  1. Series of 15N HSQCs with time delays - NMR amide exchange experiments are collected as a means of determining the presence/stability of secondary structure elements in a protein.  A typical 15N HSQC is collected (see 1d) and then the NMR sample is dried (typically two times) in order to remove the water from the sample. At this point the sample is reconstituted in D2O so that the amide protons exchange with deuterons from the solvent. A series of HSQCs is collected as quickly as possible and the amount of time for each amide proton to exchange is evaluated.  Simply put, protons that take more time to exchange are in regions of the protein containing stable secondary structural elements.

NMR structure – heteronuclear (SBNS_HT)

  1. Experiments 4a-i  
  2. 4D CC NOESY - 4D NOESY experiment (see 1c) that provides correlations between the protons attached to one carbon and that of another. Specifically, this experiment enables the user to determine which protons are nearby in space to any other carbon-attached proton in the protein.  This experiment is useful in assigning long-range distance restraints – which is critical in high-resolution 3D NMR structure determination.
  3. 4D CN NOESY - 4D NOESY experiment (see 1c) that provides correlations between the amide proton and all of the nearby carbon-attached protons. This is useful in verifying chemical shift assignments and in the determination of secondary structural elements within a protein.

NMR structure – homonuclear (SBNS_HO)

  1. Experiments for 1a-e

NMR structure – Residual Dipolar Couplings (RDCs) (SBND)

For general information regarding RDC methods/theory see this website. 

  1. IPAP 15N HSQC
  2. HNCO IPAP for Hα-Cα bonds
  3. HNCO IPAP for C’ - Cα

NMR relaxation/dynamics study (SBNR)

A series of experiments collected in order to determine the motion that exists within the molecules of interest.

  1. T1
  2. T2
  3. NOE
  4. T1þ