Final Activity Report Summary - AMYLOID NMR STUDIES (Study of amyloid formation by means of solid and liquid State NMR)
Spectroscopy deals with the interactions between electromagnetic radiation and matter. How the matter is influenced depends on the wavelength or frequency of the radiation. Nuclear magnetic resonance (NMR) is a special branch of spectroscopy, exploiting the magnetic properties of atomic nuclei. The method functions as follows: Firstly, a substance is placed in a magnetic field. Most atomic nuclei, such as hydrogen nuclei, then behave like microscopic compass needles, called nuclear spins. Each nuclear spin orientation corresponds to a different energy level. The spins may jump between the levels when the sample is exposed to radio waves whose frequency exactly matches the energy spacing. This is called resonance.
The utility of NMR in chemistry is that signals can be used to determine the number and type of chemical groups in a compound. But NMR can also provide information about the relative distances between different nuclei and in solid materials their orientation in space. The accumulated information from different NMR experiments often provides a detailed picture of the molecular structure. The complete three-dimensional structure of many proteins and other biological macromolecules has been determined in this way. However, despite its huge success, NMR is still a relatively insensitive method. This becomes particularly true for elements other than hydrogen, where the level splitting is much lower in general, the percentage of NMR active isotopes can be very low, and the specific (quadrupolar) interactions lead to very broad lines in the spectra. To get go results for biologically relevant material samples have to be synthesised and enriched with specific types of nuclear species to get a good NMR response. This is for example the case for amyloidegenic peptides.
Amyloidosis, or protein misfolding disease, is a general term describing several feared diseases associated with amyloid formation such as Alzheimer's disease, Parkinson's disease, Creutzfeld Jacob and mad cow diseases and type-2 diabetes. The detailed molecular three-dimensional structure of amyloid fibrils has been extensively investigated; nevertheless, many questions remain. In particular, it has been found that amyloid fibrils have to mature. The longer the maturation process the more homogeneous the fibrils look in electron microscopy pictures. The aim of this project was to develop an NMR methodology and implement it in order study the heterogeneity of amyloid fibrils in model systems of highly amyloidegenic peptides.
Given the above described shortcomings of NMR this project concentrated on the development of equipment and techniques that would allow structural investigations of such materials without specific isotope enrichment. The study of nitrogen-14 (14N) nuclei was addressed, since they occurred in large abundance in proteins and were involved in a set of NMR interactions capable of generating vital structural information. The problem with 14N NMR was, however, that it gave an extremely broad NMR response because it was a so-called quadrupolar nucleus of spin 1. This could be remedied by exciting a spin level transition that was in principle forbidden but could still be excited provided that sufficiently high radio-frequency field intensity was available.
For this project, a high field magnetic resonance probe for this so-called overtone spectroscopy was successfully developed. Furthermore, a software package was written allowing for numerical evaluation of the experimental results. A comprehensive set of experimental results using model systems was acquired and theoretical simulations were performed to describe the experimental results. The outcome of this assessment was that overtone NMR was less effective than originally anticipated for the study of biologically relevant materials. Alternative strategies which were developed for studying 14N nuclei had to be further explored for their applicability to amyloidal proteins and related structures.
The utility of NMR in chemistry is that signals can be used to determine the number and type of chemical groups in a compound. But NMR can also provide information about the relative distances between different nuclei and in solid materials their orientation in space. The accumulated information from different NMR experiments often provides a detailed picture of the molecular structure. The complete three-dimensional structure of many proteins and other biological macromolecules has been determined in this way. However, despite its huge success, NMR is still a relatively insensitive method. This becomes particularly true for elements other than hydrogen, where the level splitting is much lower in general, the percentage of NMR active isotopes can be very low, and the specific (quadrupolar) interactions lead to very broad lines in the spectra. To get go results for biologically relevant material samples have to be synthesised and enriched with specific types of nuclear species to get a good NMR response. This is for example the case for amyloidegenic peptides.
Amyloidosis, or protein misfolding disease, is a general term describing several feared diseases associated with amyloid formation such as Alzheimer's disease, Parkinson's disease, Creutzfeld Jacob and mad cow diseases and type-2 diabetes. The detailed molecular three-dimensional structure of amyloid fibrils has been extensively investigated; nevertheless, many questions remain. In particular, it has been found that amyloid fibrils have to mature. The longer the maturation process the more homogeneous the fibrils look in electron microscopy pictures. The aim of this project was to develop an NMR methodology and implement it in order study the heterogeneity of amyloid fibrils in model systems of highly amyloidegenic peptides.
Given the above described shortcomings of NMR this project concentrated on the development of equipment and techniques that would allow structural investigations of such materials without specific isotope enrichment. The study of nitrogen-14 (14N) nuclei was addressed, since they occurred in large abundance in proteins and were involved in a set of NMR interactions capable of generating vital structural information. The problem with 14N NMR was, however, that it gave an extremely broad NMR response because it was a so-called quadrupolar nucleus of spin 1. This could be remedied by exciting a spin level transition that was in principle forbidden but could still be excited provided that sufficiently high radio-frequency field intensity was available.
For this project, a high field magnetic resonance probe for this so-called overtone spectroscopy was successfully developed. Furthermore, a software package was written allowing for numerical evaluation of the experimental results. A comprehensive set of experimental results using model systems was acquired and theoretical simulations were performed to describe the experimental results. The outcome of this assessment was that overtone NMR was less effective than originally anticipated for the study of biologically relevant materials. Alternative strategies which were developed for studying 14N nuclei had to be further explored for their applicability to amyloidal proteins and related structures.