Abstract
Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial carriers that mimic the native bilayer environment allows for the handling of membrane proteins in solution and enables the use of small-angle scattering techniques for fast and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly non-trivial fashion, making subsequent data analysis challenging.
This thesis presents the development of a specifically deuterated, stealth nanodisc
system which can be used for SANS structural analysis of membrane proteins in solution. In combination with the D2O/H2O-based contrast variation method it is demonstrated that it is possible to prepare specifically deuterated analogues of the nanodisc, which give minimal contribution to the neutron scattering data when used in 100% D2O. An important challenge in the project was obtaining selective partial deuteration of the nanodisc system necessary for the total matching at 100% D2O. This was achieved through an E. coli based biosynthesis for both deuterated phosphatidylcholines as well as membrane scaffolding protein. To obtain physiologically relevant deuterated phosphatidylcholine (PC) species with the required scattering length density a novel method for deuteration of PC was developed to separately control the deuteration levels of three different parts of the phospholipid molecule: the lipid head-group, glycerolbackbone and fatty-acyl tails. This could be achieved via a biosynthetic pathway in a genetically modified E. coli strain adapted to growth in D2O in combination with a systematic supplementation with deuterated nutrients.
The stealth discs produced in this way should be generally usable in low-resolution
structural studes of many membrane proteins and their complexes in solution as the
analysis of SANS data for this platform is greatly simplified and allows for the
application of existing data analysis tools already available for soluble proteins
This thesis presents the development of a specifically deuterated, stealth nanodisc
system which can be used for SANS structural analysis of membrane proteins in solution. In combination with the D2O/H2O-based contrast variation method it is demonstrated that it is possible to prepare specifically deuterated analogues of the nanodisc, which give minimal contribution to the neutron scattering data when used in 100% D2O. An important challenge in the project was obtaining selective partial deuteration of the nanodisc system necessary for the total matching at 100% D2O. This was achieved through an E. coli based biosynthesis for both deuterated phosphatidylcholines as well as membrane scaffolding protein. To obtain physiologically relevant deuterated phosphatidylcholine (PC) species with the required scattering length density a novel method for deuteration of PC was developed to separately control the deuteration levels of three different parts of the phospholipid molecule: the lipid head-group, glycerolbackbone and fatty-acyl tails. This could be achieved via a biosynthetic pathway in a genetically modified E. coli strain adapted to growth in D2O in combination with a systematic supplementation with deuterated nutrients.
The stealth discs produced in this way should be generally usable in low-resolution
structural studes of many membrane proteins and their complexes in solution as the
analysis of SANS data for this platform is greatly simplified and allows for the
application of existing data analysis tools already available for soluble proteins
| Original language | English |
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| Publisher | The Niels Bohr Institute, Faculty of Science, University of Copenhagen |
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| Number of pages | 196 |
| Publication status | Published - 2014 |