Molecular Properties of Water-Soluble Chlorophyll Protein: Implications for its Biological Function
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Abstract
Water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are unusual chlorophyll (Chl)-binding proteins in several respects. While all other Chl-binding proteins of higher plants are membrane bound and contain a large number of Chls as well as carotenoids, WSCPs are water-soluble and bind a limited number of Chl molecules and no carotenoids. Their biological function remains enigmatic, although functions as protease inhibitor, as Chl and/or Chl derivative carrier, and as a source of reactive oxygen species have been proposed.
In this thesis, a potential function as protease inhibitor was investigated using recombinant WSCP from Arabidopsis thaliana (AtWSCP), which reportedly inhibits papain-like cysteine proteases. However, the misfolding of WSCP apoprotein made it impossible to identify the inhibitory domain of AtWSCP and to describe the molecular mechanisms of protease inhibition.
Furthermore, the interaction between WSCP and the thylakoid membrane was investigated, from which WSCP apoprotein can extract Chl molecules. Slow and inefficient extraction of Chl from the thylakoid membrane by WSCP apoprotein suggests that WSCP apoprotein does not directly interact with Chl-binding proteins, which are embedded in the thylakoid membrane and bind the vast majority of the Chl molecules in the thylakoid membrane. More likely, WSCP apoprotein interacts directly with lipid bilayers that contain the thylakoid membrane lipids MGDG, DGDG or PG, and can extract Chl from those, whereas it does not interact with PC membranes. WSCP-Chl complexes are not able to interact with membranes anymore, suggesting that a ring of hydrophobic amino acids with two conserved Trp residues around the Chl binding site plays a central role in the WSCP-membrane interaction and in the Chl uptake by WSCP.
In addition to the Chl binding from membranes, the tetrapyrrole specificity of various versions of WSCP was studied in the present work. Titrations and time-resolved CD measurements allowed to determine KD values and kinetic parameters for the binding of Chl a/b, chlorophyllide a/b and pheophytin a/b. The Chl a/b selectivity in WSCP is thermodynamically controlled. Chl b binding is preferred when a hydrogen bond can be formed between the C7 formyl of the chlorine macrocycle and the protein, whereas Chl a is preferred when Chl b binding is sterically unfavorable. Overall, Chl is bound with higher affinities than chlorophyllide or pheophytin, which indicates that the phytol chain and the central Mg2+ ion are important interaction sites between WSCP and tetrapyrrole. Pheophorbide, which lacks both phytol chain and the central Mg2+ ion, can only be bound as pheophorbide b to a WSCP, which has a higher affinity for Chl b than Chl a. This suggests that the hydrogen bond between WSCP and C7 formyl is another important interaction for tetrapyrrole binding to this WSCP. Moreover, WSCP was able to bind the porphyrins protochlorophyllide and Mg-protoporphyrine IX, indicating that the size of the π electron system of the macrocycle and the presence of a fifth ring at the macrocycle do not noticeably affect the binding to WSCP. Surprisingly, also heme can be bound to WSCP, which suggests that the type of central ion is insignificant for the WSCP-tetrapyrrole interaction.
Finally, the potential of WSCP-bacteriochlorophyll (BChl) complexes for an application as photosensitizer in photodynamic therapy (PDT) was explored in this thesis. PDT makes use of photosensitizers that upon illumination produce reactive oxygen species like singlet oxygen in order to induce cell death in tumor cells or pathogenic microorganisms. The WSCP-BChl complex exhibits several properties that are advantageous for an application in PDT. The complex exhibits a strong light absorption at 770 – 780 nm, where light penetration into tissue is maximal, and produces considerable amounts of singlet oxygen upon illumination, but also shows a high photo and heat stability, which makes WSCP-BChl an interesting candidate for usage in PDT.