Nuclear magnetic resonance studies of growth hormone releasing factor analogs.

• Main Author: Lamb, Michael Paul.
• Other Authors: MacKenzie, Neil
• Language: en
• Published: The University of Arizona. 1996
• Online Access: http://hdl.handle.net/10150/187507

The solution structures of three potent growth hormone releasing factor (GRF) analogs corresponding to GRF(1-29), GRF(1-44), and GRF(1-76) were investigated by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies. The analogs contained a deletion of Tyr$\sp1$ and acylation of Ala$\sp2$ with a para-methyl hippuryl group. In addition, eight amino acid substitutions were made in the GRF(1-76)OH analog versus human GRF. The substitutions were retained in the respective shorter analogs. Each of the three peptides (pmh29, pmh44, and pmh76, for simplicity) was studied in aqueous solutions of trifluoroethanol (TFE), while pmh76 was additionally studied in an aqueous buffer. Complete sequential assignment was made for pmh29 in 45% TFE. Nuclear Overhauser effect (NOE) data, chemical shift data. and CD spectra defined pmh29 as nearly completely helical. Long-range NOEs in the NOESY spectra of pmh29 revealed the presence of tertiary structure. Split proton resonances and a wide span of long-range NOE interactions suggested a conformationally heterogeneous tertiary structure. Distance geometry and simulated annealing were performed using NOE data to calculate the two predominant tertiary structures. The calculated structures for pmh29 were in accordance with a consensus of previously proposed bioactive conformational characteristics. Complete sequential assignment was accomplished for pmh44 in 45% TFE. The pmh44 molecule was nearly completely helical as determined by NOE data, chemical shift data. and CD spectroscopy. Long-range NOEs provided evidence of tertiary structure in the pmh44 molecule. The coincidence of chemical shifts and long-range NOEs in pmh44 and pmh29 pointed to a similar structure for both molecules, with the 15 C-terminal residues of pmh44 forming a linear helical extension. Severe signal overlap impaired the assignment of pmh76 in 30% TFE and aqueous buffer. Chemical shift correlation between the three analogs in TFE suggested a similar conformation in the corresponding regions of the pmh76 peptide, with the C-terminal extension forming a linear helix. These studies provide the first evidence of a global tertiary fold in any GRF analog or member of the secretin family. The potency of these analogs is likely due to a combination of metabolic stability and the adoption of preferred secondary and tertiary structures.