1. Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements
Journal of Biomolecular NMR, 16: , 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 NPY28 32
Lactam bridge
Biochimica et Biophysica Acta 1435 (1999)

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

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

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

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 NMRGz

9. Measurement diffusion coefficient: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)
g and  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
2 and 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 be known

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. Association is concentration independent !
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
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