Method: Peptides were synthesised using solid-phase Fmoc chemistry, purified by high performance liquid chromatography and ~1.0 mM solutions made up in phosphate buffer (PB) at pH 7.4. NMR sample composition consisted of PB:D20 (90:10 v/v) at 303K. 1H-NMR and Diffusion Ordered Spectroscopy (DOSY) spectra were obtained using a Bruker AMX-600, and TOtal Correlation Spectroscopy (TOCSY) and Nuclear Overhauser Effect (NOE) spectra using a Bruker AB-400.
Results: Histatin-1 chemical shifts for peptide backbone and side-chain protons were identified using the TOCSY and NOE spectra. Lack of long range NOE signals across the molecule and fast 1H/2H amide exchange indicated a flexible, and essentially random unstructured conformation of histatin-1. This finding is in keeping with structures found for the lower molecular weight stuctural analogues histatin-3 and histatin-5, which only have low affinity HAP binding. The aromatic regions of the proton spectra of histatin-1 and statherin showed broad and poorly resolved backbone amide signals indicative of high flexibility, and water solvation. However, the proton spectrum of the mixture was very different; 17 out of 81 amide signals were dramatically shifted downfield forming a new sharp narrow multiplet indicative of slow exchanging and buried amide protons. The 2D-DOSY experiment yielded diffusion coefficients confirming this interaction.
Conclusion: This NMR study suggests protein-protein interactions between the high affinity HAP proteins histatin-1 and statherin.
We acknowledge support of a Heptagon Fund Technology Validation Award.