Peptide Structural Analysis Exercise

This exercise will illustrate practical aspects of polypeptide structural analysis including solubility, conformational averaging, and proton NMR resonance assignment.

Subject: A 15-residue peptide corresponding to one of seventeen overlapping segments of the human Hsp10 (left-overs from Dr. Landry's immunology project, see below).
 

Tasks

  1. Peptide solubilization - Weigh out approximately 1 mg peptide and attempt to dissolve it in 50 microliters of water (final concentration, approximately 2 mM); failing that, then add 10-30% hexafluoroisopropanol (HFIP); failing that, then prepare a sample in DMSO.  [Schedule approximately 1 hour with Landry-lab analytical balance and pipettemen?]  Check sample pH using indicator paper.
  2. Conformational analysis by CD - Obtain far-UV CD spectra of 50 micromolar samples in 10 mM phosphate buffer, buffer plus 10% HFIP, buffer plus 20% HFIP, and buffer plus 30% HFIP.  [Schedule 1 hour with instructor or designee and CD spectropolarimeter]
  3. Prepare 0.7 ml NMR sample of 2 mM peptide in buffer plus 30% HFIP-d2 (D, 98%).  Obtain 2D proton NMR spectra for assignment of backbone resonances, nuclear Overhauser effect (NOESY) and total correlated (TOCSY) spectra.  Students will be encouraged to observe data acquisition and processing by the instructor or NMR facility manager.  All spectra should be completed by mid-semester.  Data plotting and assignment to be carried out by students using the PC called MrBlack.
  4. Project report - CD spectra with interpretation (fraction alpha helix), table of backbone proton NMR assignments and annotated fingerprint (NH-alpha) and NH-upfield regions of the TOCSY spectrum with interpretation (conformational behavior, e.g., deviations from random coil values)

    5.  
Each peptide has a high likelihood of solubility in water.  The peptides are expected to be random coils in water but have varying degrees of alpha helix in HFIP.  Some peptides have as many as three of the same residue type, thus necessitating methods of sequential assignment.  Several peptides have proline, which will exist in cis and trans conformers.  Thus, two sets of resonances will be present for several residues surrounding the proline.  This is a good illustration of conformational heterogeneity.  Since these are overlapping peptides, some subsequences can be shared by two peptides, potentially revealing effects of context on chemical shift.  Results will be exchanged among students and discussed in class.

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