Department of Biochemistry, University of Sydney 2006, Australia and *Garvan Institute of Medical Research, Sydney 2010, Australia
The neuropeptide galanin is widely distributed in the central and peripheral nervous systems of the human and other animals. The peptide is involved in a wide range of cellular and physiological events including modulation of the secretion of a number of hormones and neurotransmitters, various effects on the cardiac and other muscle systems, suppression of pain, sexual activity, learning and memory, and stimulation of appetite.
We have investigated the structural properties of the 30-residue wild-type human galanin (hGAL) and shown, by two-dimensional NMR spectroscopy, that it forms a range of `nascent structures' in aqueous solution which are in rapid equilibrium with random coil conformations. In particular, residues 3-12 form a `nascent helix'. Nascent helix has a strong propensity to form regular helix if it can be appropriately stabilized. This can be achieved in vitro by adding a helix-promoting solvent such as trifluoroethanol (TFE). The nascent helix includes a hydrophobic core centred on Tyr9; the side-chain protons of Tyr9 interact with the side-chain protons of Ser6, Ala7, Leu10 and Leu11 a significant proportion of the time.
The importance of the N-terminal half of galanin is reflected in the observations that it is strictly conserved across mammalian and many other species and that, with only a few exceptions, is required for receptor binding and agonist activity. We speculate, based on the properties of nascent structures in other peptides, that the nascent structures in hGAL act as areas of recognition for the galanin receptor and promote folding of the peptide once galanin is bound to the receptor.
We have examined the structural and functional properties of two hGAL mutants. A `super' agonist, 8Gly->D-Trp, which binds to galanin receptor with an affinity three orders of magnitude greater than that of wild-type hGAL, and a `weak' agonist, 9Tyr->Ala, which binds to receptor with an affinity three orders of magnitude lower than that of wild-type. We predicted that the super agonist would have a more stable hydrophobic core with respect to the wild-type peptide and therefore would contain a larger percentage of stable structure over a range of solution conditions such as temperature, and the presence and absence of TFE and the `crowding' agent glycerol. In contrast, the Ala9 analogue would have a less stable hydrophobic core than the wild-type and would possess less structure over the same range of conditions.
Circular dichroism spectropolarimetry has confirmed these hypotheses and, in addition, has indicated that the propensity for self-association of all three peptides in high concentrations of TFE (>70%) follows the order [D-Trp8]hGAL > hGAL > [Ala9]hGAL. This order is consistent with the hydrophobic core acting as the binding site for self-association.