Perdeuteration of Biological Macromolecules: A Case Study of Human Carbonic Anhydrases

Katarina Koruza

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

194 Nedladdningar (Pure)

Sammanfattning

Deuterated biomolecules - where hydrogen atoms (H) are exchanged to its isotope deuterium (D) - are essential for biological experiments in neutron scattering, but they are not readily available. In neutron scattering, D has a strong positive coherent scattering length (6.67 fm) in comparison with the negative scattering length of H (-3.74 fm). These different characteristics of neutrons to D and H are used in neutron protein crystallography (NPX) where the use of deuterated proteins maximizes signal-to-noise ratio, reduces background and gives better nuclear scattering length density maps. At the same time, NPX allows at resolutions of ∽2.5 Å or better to unambiguously determine the positions of the exchanged D. This is important because H atoms constitute nearly half of all the atoms in a protein molecule. Accurate structural information about H positions and hydrogen bonds that are involved in most aspects of protein structure and function remains challenging by using X-ray crystallography alone. Therefore, NPX offers a unique and complementary approach to X-rays for locating H atoms. My thesis focuses on production of recombinant proteins for NPX with human carbonic anhydrase IX (hCA IX) as the case study protein. The long-term ambition of my work is to show that joint X-ray crystallography and NPX can provide insight into protein–ligand interactions and can give valuable input into structure-guided drug design and computational chemistry. As an example, for this, an emerging cancer target hCA IX was chosen, that is in need of the development of isoform-specific inhibitors. In paper I, we demonstrate the improvement of binding affinity of the inhibitor acetazolamide to hCA IX when conjugated to a polypeptide. In paper IV, we discuss crystallisation optimisation for large crystal growth, that is often a bottleneck for NPX experiments. Finally, in paper IV we describe jointly refined X-ray and neutron crystal structures of hCA IXmimic (hCA II with mutations that mimic the active site of hCA IX) with two selective inhibitors. This study shows how neutron studies can contribute unique information on inhibitor binding. I was particularly interested in improvement of (per)deuteration in living organisms in order to maximize recombinant protein production and expression of hCA IX under deuterium stress, which are the main discussion points of paper II and III. In paper II, we address some of the technical difficulties of protein deuteration in vivo in bacteria, and study the biophysical effects of deuteration on the protein product. Further on we explore the possibilities improvement of deuteration of proteins by supplementing a minimal growth media with a fully perdeuterated extract from algae, as described in paper III.
Originalspråkengelska
KvalifikationDoktor
Tilldelande institution
  • Biologiska institutionen
Handledare
  • Knecht, Wolfgang, handledare
  • Fisher, Zoë , Biträdande handledare
  • von Wachenfeldt, Claes, Biträdande handledare
Tilldelningsdatum2019 maj 24
UtgivningsortLund
Förlag
ISBN (tryckt)978-91-7895-068-3
ISBN (elektroniskt)978-91-7895-069-0
StatusPublished - 2019

Bibliografisk information

Defence details
Date: 2019-05-24
Time: 13:00
Place: Biologihörsalen, Biologihus A, Sölvegatan 35, Lund
External reviewer(s)
Name: Lundqvist, Tomas
Title: Dr.
Affiliation: RISE Research Institutes of Sweden
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Ämnesklassifikation (UKÄ)

  • Biokemi och molekylärbiologi

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