D2O NMR Studies on Packed Poly-proline

Contents

0.1  Introduction  2

0.2  Biological Motivation  2

0.3  Physical Background  3

0.3.1  Quadrupolar Interaction  3

0.3.2  Quadrupolar Interaction for Deuterium  4

0.3.3  Interpretation of Powder Pattern  5

0.4  Materials  5

0.4.1  Poly-proline  5

0.4.2  Attachment System  6

0.5  Methods  6

0.5.1  Preparing of Poly-proline  6

0.5.2  NMR Assignment  7

0.6  Results and Analysis  7

0.6.1  Nonlinear Fit of LS2 Spectra  8

0.7  Discussion  9

0.8  Conclusion  10

  Bibliography  11

research project is, to investigate this

Although solid state D (deuterium) NMR (nuclear agnetic resonance) has often been used for structural

assumption.

investigations of proteins and polymers under different

0.2 Biological Motivation

hydration levels or temperatures, this has never been done under different densities of polymer. Therefore the

The origin of life is a controversially discussed D NMR spectra (also called Pake or powder pattern) of

field in the scientific community with many

poly-proline (PP) - D2O mixture was analyzed as a hypotheses [2]. However most models agree that

function of different packing magnitudes, applied by a amino acids and nucleotides were present at the

plunger-screw system. By analyzing the data it was possible to assume that the higher packing of poly-

beginning and condensed nonenzymatically to proline diminished the motion of the water inside

form oligomeric products. Yet the mechanism the polymer chains.

how this happened is still discussed, Leman et Factors defining the three dimensional

al. have proposed a theory in which the organization of proteins are of scientific interest,

polymerization took place in an aqueous solution because their function is mainly dependent on

under the presence of carbonyl sulfide, a simple the folding of the amino acid chain. One of these

volcanic gas [3]. Therefore it is possible that the factors, besides the amino acid sequence,

first biological polymers, emerged near deep sea temperature or pH level, seems to be the

volcanoes and ignited the evolution of life. packing magnitude of the protein. Investigation

Another recent model has been published by of this influence on the D NMR (also referred as

Gold et al., which sees indications that microbial 2H NMR) spectra will be the topic of this paper.

life emerged in the crust of earth [4].

0.1 Introduction

It is clear that under these circumstances, deep sea or underground, pressure is much higher

Studies have shown, that poly-proline shows than under our biosphere. Therefore it is

indications for formation of two different binding interesting to investigate the structural behaviour

sites for water [1]. The weaker one exists of poly-amides, like poly-proline, under

between water and the majority of the proline differentpacking magnitudes. This could lead to a

protons and could represent the interactions of better understanding of the conformational

the surrounding water and the protein shell. The behaviour of polymers under different packings.

different spin states with different energies, also

Figure 1: Possible water molecule binding site on the referred as spin up and spin down and can be

poly-proline molecule in its helical conformation [1] second one shows very strong spin-spin

interpreted as magnetic dipoles. This couplings between the water protons and a small

quantization of spins is described by the fraction of the proline proton populations and

following equation are:

could be due to the presence of helical structures acting as a hydrogen-bonding linker If the external magnetic field B0 is applied the

between the helices elements O1, C_1 and C_2 dipoles orient only parallel or antiparallel to it.

(see Figure 1). Recent investigations of poly- The energies of different spin states:

proline in this laboratory have shown anomalous T1 and T2 hydration dependences, which has been interpreted in terms of a cooperative water

In a case of a spin 1/2 system the energy molecule - PP molecule helical conformational

difference is _E = ~B0. The gyromagnetic ratio is rearrangement. During this work they also

a different constant for every nuclei. If the observed that the magnitude of packing of poly-

magnetic field B0 is held constant, B0 and is Proline was influencing the NMR spectra. By

abbreviated by −!0, which is called the Larmor applying different pressure during the placement

frequency. The difference between the numbers of the polymer into the NMR tube the NMR

of spins in the spin up and down state is small spectra changed. This underlines the possibility

and can be described by the Boltzmann that an outside force could influence the

distribution:

structure of the peptide. The goal of this

for a hydrogen nuclei with I = 1/2, B0 = 11 T and The different energy levels would have the same

room temperature. This small imbalance gives distance without the quadrupolar interaction

rise to a small polarization of the nuclear spin in between the nuclei (eQ·eq=e2qQ), so only one

the sample [5]. NMR experiments are performed absorption line would be measureable (see

with a high field superconductive magnet that Singulett in Figure 3). As a results of

produces a magnetic field B0, which is thequadrupolar interaction the energy levels for

homogeneous, strong and orient the spin either mI = ±1 and mI = 0 are altered and the distances

up or down. A coil produces an electromagnetic between them are no more the same. This

waves also referred as B1, but which is also able allows 3

to measure the magnetization of the sample. The

by intramolecular nuclear quadrupole around the previous frequency _0 for the

interactions between the quadrupole moment singulet. For nuclei with I_1 the quadrupolar

and the electric field tensor. This is also called interaction is the dominating relaxation

electrical quadrupole moment mechanism. Because the relaxation times TQ 1

are very fast, the width of the spectra are big. (e electron charge, _ charge density, r distance Therefore most quadrupolar nuclei are not of volumelemet d_ from center, _ angle between favoured for high resolution NMR spectroscopy. radius vector r and axis of spin, Q deuteron Deuterium is the exception with a relative small quadrupolar moment) and gives us an idea quadrupolar moment Q [10−28m2] = 2.8 · 10−3 about the spherical symmetry of the positive compared to e.i. 14N 1.7·10−2.

charge distribution. As a consequence the nuclei

0.3.2 Quadrupolar Interaction for

not only interact with B0 of the applied magnetic

Deuterium

field but also with the local electrical field. Many The D-NMR spectra show two resonance lines s

nuclei experience an unsymmetrical distribution explained in the previous paragraph, the

of their electrons, and therefore are in an difference between the two is referenced as

inhomogeneous electrical field with the electrical quadrupolar splitting __Q. The component Vzz is

field gradient (EFG) Vzz = e · q = (@2V/@z2). In the biggest element of the electrical field gradient

this field gradient the quadrupolar nuclei can only in diagonal form. _ = (Vxx −Vyy)/Vzz defines the

align along certain orientations, which represent assymmetrical parameter of the electrical field

specific energies. The spin I= 1 can only have gradient (|Vzz| _ |Vyy| _ |Vxx|, 0 _ _ _ 1). When Vzz

three orientation in a B0-field, which correspond is parallel relative to the magnetic field the

to the magnetical quantum numbers mI = +1, quadrupolar splitting is described by the

0,−1. The different energy levels Em I can be following equation: