D2O NMR Studies on Packed Poly-proline

Lorenz Steinbock

January 14, 2006

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

Although solid state D (deuterium) NMR (nuclear agnetic resonance) has often been used for structural investigations of proteins and polymers under different hydration levels or temperatures, this has never been done under different densities of polymer.Therefore the D NMR spectra (also called Pake or powder pattern) of poly-proline (PP) - D2O mixture was analyzed as a function of different packing magnitudes, applied by a plunger-screw system.By analyzing the data it was possible to assume that the higher packing of polyproline diminished the motion of the water inside the polymer chains.

Factors defining the three dimensional organization of proteins are of scientific interest, because their function is mainly dependent on the folding of the amino acid chain.One of these factors, besides the amino acid sequence, temperature or pH level, seems to be the packing magnitude of the protein.Investigation of this influence on the D NMR (also referred as 2H NMR) spectra will be the topic of this paper.

0.1 Introduction

Studies have shown, that poly-proline shows indications for formation of two different binding sites for water [1].The weaker one exists between water and the majority of the proline protons and could represent the interactions of the surrounding water and the protein shell.The second one shows very strong spin-spin couplings between the water protons and a small fraction of the proline proton populations and could be due to the presence of helical structures acting as a hydrogen-bonding linker between the helices elements O1, C_1 and C_2 (see Figure 1).Recent investigations of polyproline in this laboratory have shown anomalous T1 and T2 hydration dependences, which has been interpreted in terms of a cooperative water molecule - PP molecule helical conformational rearrangement.During this work they also observed that the magnitude of packing of poly- Proline was influencing the NMR spectra.By applying different pressure during the placement of the polymer into the NMR tube the NMR spectra changed.This underlines the possibility that an outside force could influence the structure of the peptide.The goal of this research project is, to investigate this assumption.

Figure 1: Possible water molecule binding site on the poly-proline molecule in its helical conformation [1] -- in downloadfile

0.2 Biological Motivation

The origin of life is a controversially discussed field in the scientific community with many hypotheses [2].However most models agree that amino acids and nucleotides were present at the beginning and condensed nonenzymatically to form oligomeric products.Yet the mechanism how this happened is still discussed, Leman et al.have proposed a theory in which the polymerization took place in an aqueous solution under the presence of carbonyl sulfide, a simple volcanic gas [3].Therefore it is possible that the first biological polymers, emerged near deep sea volcanoes and ignited the evolution of life.Another recent model has been published by Gold et al., which sees indications that microbial life emerged in the crust of earth [4].It is clear that under these circumstances, deep sea or underground, pressure is much higher than under our biosphere.Therefore it is interesting to investigate the structural behaviour of polyamides, like poly-proline, under different packings.

0.3 Physical Background

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[1] J. A. Stanley and H. Peemoeller, Characterization of polyproline-water system by NMR spin, 1991, J. Phys. II France, 1, 1491-1503

[2] http://en.wikipedia.org/wiki/Origin of life

[3] Luke Leman, Leslie Orgel, M. Reza Ghadiri1, Carbonyl Sulfide–Mediated Prebiotic Formation of Peptides, 2004, Science, 306, 283-286

[4] Thomas Gold, The Deep, Hot Biosphere, 1992, Proc. Natl. Acad. Sci., 89, 6045-6049