NMR Solution Structure of C-terminally amidated, Full-length Human Galanin
The neuropeptide galanin adopts an irregular secondary structure.
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
Chemical shift data for Ac-hGal(17-30)NH2, a C-terminal Galanin fragment
NMR solution structure for Ac-hGal(17-30)NH2, a C-terminal Galanin fragment
Chemical shift data for hGal(2-12)KK, a galanin N-terminal fragment
NMR solution structure for hGal(2-12)KK, a galanin N-terminal fragment
Chemical shift data for hGal(1-12)KK, a galanin N-terminal fragment
NMR solution structure for hGal(1-12)KK, a galanin N-terminal fragment
Chemical shift data for hGal(1-17)KK, a galanin N-terminal fragment
The authors state the following regarding the geometric deviations with this ensemble: With most of the hydrogen bonds in the N-terminus of the peptide bifurcated, or even more, they are confident that the ensemble represents the time-averaged structure of a peptide that is rapidly interconverting turns of alpha, 310-, and pi-helix. Their shift assignments were largely unambiguous and can be found in BMRB entry 31032. The "irregular helix restraints" used (and described in the manuscript and its references) were employed to allow the ensemble to sample multiple arrangements during simulated annealing. By definition, this ensemble existing as an average will display a significant degree of geometric irregularity, particularly with regard the backbone and sidechain outliers as compared to statistical norms. Further, the clashscore level is quite high. This appears to be a consequence of the very high density of ROE-derived distance restraints in the N-terminus of the peptide, which were maximally relaxed to account for any uncertainty in their measurement throughout several rounds of validation and re-calculation. Tight sidechain packing and compression seems to be the dictated outcome of our data. Much of their study is concerned with compact hydrophobic packing in this sequence - this full-length peptide is indeed much more rigid across the stretch of residues it shares with fragment peptides described in entries 7S3O, 7S3Q, and 7S3R. Together with the compactness of the region of the sequence, the clashes appear to be a consequence of several averaged, coiling, helical conformers all existing on the NMR timescale. This is discussed at further length in the citation affiliated with this deposition
sample was dissolved in 10 mM sodium acetate-d3/acetic acid-d4 buffer at pH 4.6; no additional additives were present.
0.5 mM hGal(1-30)NH2, 4.14 mM [U-2H] sodium acetate, 5.86 mM [U-2H] acetic acid, 95% H2O/5% D2O
peptide was suspended and dissolved into deuterated acetate buffer, which had been prepared in 95:5 H2O:D2O to within 0.02 units of pH 4.6
Brunger, Adams, Clore, Gros, Nilges and Read
MestreLab (Mnova / MestReNova / MestReC)
Peptide concentration was assessed over a fifty-fold range of concentrations in 1D 1H NMR spectra from 0.1 mM up to 5.0 mM. While all concentrations below and inclusive of 1.0 mM peptide were acceptable - free of dispersion and broadening effects across multiple weeks in solution - 0.5 mM was selected as the optimal choice for further analysis with optimal resolution.