Journal of Biomolecular NMR, 16 : 109-119, 2000.

1. Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements Journal of Biomolecular NMR, 16: 109-119, 2000.

2. Introduction • NPY- Neuropeptide Y • The C-terminal region of NPY 13-residue, • C-terminally amidated. • Polypeptide hormone and neurotransmitter, • active in both the central and nervous systems. • It participates in the regulation of many • physiological processes, including food intake, • blood pressure, etc.

3. The amino acid sequence of NPY 28 32 Lactam bridge Biochimica et Biophysica Acta 1435 (1999) 127-137

4. Stereo views of the monomer of NPY in 40% TFE Biochimica et Biophysica Acta 1435 (1999) 127-137

5. Model of dimer of NPY in 40% TFE Biochimica et Biophysica Acta 1435 (1999) 127-137

6. Model of tetramer of NPY in 40% TFE Biochimica et Biophysica Acta 1435 (1999) 127-137

7. PFG Spectroscopy • PFGNMR method allows the translation diffusion coefficient of the • molecule to be determined under identical conditions to those used • for determination of the solution structure. • The state of self-association of a protein can be obtained directly • from its diffusion coefficient or via the relationship between • its mass and diffusion coefficient. • Diffusion coefficients were measured by incrementing either the • duration of the field gradient Pulses,in which peak intensities • and volumes were fitted to a single exponential decay.

8. The pulse program of PFG NMR Gz Gz

9.  g G • Measurement diffusion coefficient: • The intensity of the NMR signal in the PFG diffusion ordered experiment is described by: • I = I0 exp(-2g2D2(- /3)) • I and I0 are the intensity of the NMR signal in the presence and absence of external gradient pulses (exp) • D is the diffusion coefficient (calculate) •  is the time period over which translational diffusion is allowed to occur (Known) •  is the nuclear gyromagnetic ratio (Known) • gand are the amplitude and duration of the gradient pulse (Known)

10. Calculation of apparent molecular mass from translational diffusion coefficient • The relationship between molecular mass (M) and diffusion coefficient (D) is given by: • M = ( k T/6FD)3[4  NA/[3(2 + 11)]] • k is the Boltzmann constant • T is the absolute temperature •  is the viscosity of the solution • NA is Avogadro’s number • 2and 1 are the partial specific volumes of the molecule and solvent water • 1 is the fractional amount of water bound to the molecule (hydration number) • F is the shape factor. • Calculation mass, M, from diffusion coefficient, D, using Equation requires the values for , 2, 1 and F to beknown

11. Diffusion coefficient in water at 20 ℃ In order to take into account the differences in temperature and viscosity among different solvents, it is convenient to convert the experimentally measured diffusion coefficients to standard conditions, usually water at 20 C: D20,w= Dobs(293.2/T)/(ηT,w /η 20,w) (η s /ηw) • D20,w is the diffusion coefficient standardised to water at 20℃ • Dobs is the measured diffusion coefficient in the actual solvent at the • experimental temperature(T) • ηT,w and η 20,w are the viscosities of water at the temperature of the • experiment (T) and at 20 ℃ • η s and ηw are the viscosities of the solvent and water at a common • temperature

12. To ensure that sample had equilibrated with respect to sample temperature and state of self-association, measurements were taken consecutively until no systematic change in the diffusion coefficients was observed. TFE NPY

13. H2O/D2O TFE Square-peptide II Circle-peptide III Association is concentration independent !

14. Diffusion coefficients of peptides I-III Exp. Average Standard

15. Mass of peptide I-III calculated from diffusion coefficients exp stand

16. Conclusion • From the molecular mass calculated from diffusion • coefficient show that the peptides are mainly monomeric • in water but associate to dimers in aqueous TFE.

17. NMR-derived structure of peptide I Lactam bridge Back