Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements

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

NPY- Neuropeptide YThe 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.

Introduction

The amino acid sequence of NPY

Biochimica et Biophysica Acta 1435 (1999) 127-137

28 32

Lactam bridge

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

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.

PFG Spectroscopy

The pulse program of PFG NMR

Gz Gz

Measurement diffusion coefficient:

The intensity of the NMR signal in the PFG diffusion ordered experiment is described by:

I = I0 exp(-2g2D2(- /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)

G

g

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/6FD)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

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 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

Diffusion coefficient in water at 20 ℃

D20,w = Dobs(293.2/T)/(ηT,w /η 20,w) (η s /ηw)

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

Association is concentration independent !

TFE

H2O/D2O

Square-peptide II Circle-peptide III

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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