Download Biochimica et Biophysica Acta 1435 (1999) 127-137

Peptide self-association in aqueous trifluoroethanol
monitored by pulsed field gradient NMR diffusion
measurements
Journal of Biomolecular NMR, 16: 109-119, 2000.
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.
The amino acid sequence of NPY
28
32
Lactam bridge
Biochimica et Biophysica Acta 1435 (1999) 127-137
Stereo views of the monomer of NPY in 40% TFE
Biochimica et Biophysica Acta 1435 (1999) 127-137
Model of dimer of NPY in 40% TFE
Biochimica et Biophysica Acta 1435 (1999) 127-137
Model of tetramer of NPY in 40% TFE
Biochimica et Biophysica Acta 1435 (1999) 127-137
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.
The pulse program of PFG NMR
Gz
Gz

Measurement diffusion coefficient:
g
G
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)
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
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
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
H2O/D2O
TFE
Square-peptide II
Circle-peptide III
Association is concentration independent !
Diffusion coefficients of peptides I-III
Exp.
Average
Standard
Mass of peptide I-III calculated from diffusion
coefficients
exp
stand
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.
NMR-derived structure of peptide I
Lactam bridge
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