1H NMR analysis and in vitro bioactivity of Leu27-bGRF(1-29)NH2 and its D-Ala2 and des-(Tyr1-Ala2)-analogs.
Kloosterman. D A DA; Scahill. T A TA; Hillman. R M RM; Cleary. D L DL; Kubiak. T M TM
Key Findings
- Leu27‑bGRF(1‑29)NH2 (II) retains about 48% of the activity of the full‑length bGRF(1‑44)NH2 (I).
- Replacing Ala2 with D‑Ala (III) raises activity to ~77% of the full‑length peptide, though the increase isn’t statistically significant.
- Removing residues 1‑2 (IV) drops potency by ~10,000‑fold, showing the N‑terminal residues are critical for receptor interaction.
- Similar helical structures in solution do not predict biological activity, indicating that shape in the test solvent isn’t the key factor.
Practical Outcomes
- For DIY peptide users, keep the first two amino acids of GRF‑1‑29 intact; dropping them makes the peptide essentially useless. A D‑Ala substitution at position 2 may slightly boost activity, but the benefit is modest. This info helps fine‑tune peptide design or selection for growth‑hormone‑releasing purposes, though real‑world effects in humans still need clinical testing.
Summary
The study compared three versions of a bovine growth‑hormone‑releasing peptide. The full‑length peptide (I) was the most active, a version missing the first two amino acids (IV) was almost dead, and a version with a D‑Ala change at position 2 (III) was almost as active as the full‑length peptide. The shape of the peptides in a test solution didn’t explain the activity differences, suggesting the very start of the molecule matters for binding to the receptor.
Abstract
Relative growth hormone-releasing potencies of bovine growth hormone-releasing factor (bGRF) analogs bGRF(1-44)NH2 (I), Leu27-bGRF(1-29)NH2 (II) and D-Ala2, Leu27-bGRF(1-29)NH2 (III) in in vitro bovine anterior pituitary cell cultures were determined to be 100%, 48% and 77%, respectively. The potencies of II and III, although numerically different, were not statistically different. Leu27-bGRF(3-29)NH2 (IV) was approximately 10,000 times less potent than 1. 1H NMR studies of peptides II, III and IV in 35% d3-2,2,2-trifluorethanol (TFE)/65% phosphate buffer at pH 4 revealed very similar, highly helical secondary structures in the 8-29 region, with only subtle differences at the N-termini. This lack of correlation between secondary structure in solution and in vitro bioactivity suggests that either 1) the biological conformations induced at the GRF receptor for II and III vs. IV are different from those generated in TFE/buffer, 2) similar secondary structures may be necessary but not sufficient for the observed bioactivity or 3) residues 1 and 2 of analogs II and III are important contact residues crucial for effective GRF-receptor interaction.
Study Information
pubmed
1991