Pharmaceutical analysis - Peptide and Protein Structure by NMR
The primary and secondary structure of small peptides (15 - 20 residues) may be determined by homonuclear experiments alone as follows:
- Acquire DQF-COSY , TOCSY, and NOESY data
- Analyze TOCSY and DQF-COSY for spin system and residue type assignment
- Analyze NOESY for sequence specific resonance assignments
- Perform chemical shift index calculations for secondary structure
- Analyze data for side-chain assignments
- Assign NOESY cross-peaks
- Perform structure calculations with simulated annealing
For larger peptides, peptides bound to proteins, or proteins, uniform 13C and 15N labeling may be required. A typical approach may include:
- Acquire 2D and 3D 15N-HSQC, 13C-HSQC, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO data
- Analyze data for 15N, 13C, and proton assignments
- Acquire 15N-filtered NOESY, 13C-filtered NOESY, 15N/ 13C-NOESY data
- Assign NOESY cross-peaks
- Perform structure calculations with simulated annealing
Another application includes chemical shift mapping of small molecule binding to labeled proteins and peptides. An 15N labeled protein, with resonances assigned as above, is "titrated" with a small molecule inhibitor candidate and 15N HSQC is acquired. Changes in the chemical shifts of the protein will reveal the regions where the small molecule binds.
We have a 500 MHz 3-channel Varian Unity Inova NMR spectrometer equipped with a triple resonance inverse probe. We have experience in solving the structure of free peptides in solution, and proteins up to 40 kDa. Please feel free to call us for all of your protein structural needs.
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