Phytoglobins: Elucidation of Structural, Functional and Practical Features of Non-Symbiotic Plant Hemoglobins

Abstract

Phytoglobins (Pgb) (plant hemoglobins) are heme-containing proteins and less well known in comparison to the mammalian counterparts hemoglobin (Hb) and
myoglobin (Mb). They share the same secondary structure as other globin-related proteins, six to eight α-helices comprising the myoglobin-fold (Mb-fold) with one prosthetic heme moiety per subunit. Pgbs are divided into different classes, where the non-symbiotic Pgbs (nsPgb) are found in vast number of plants. Unlike their mammalian counterparts, nsPgbs usually have an additional coordination site interacting with the iron called the distal histidine, in addition to the proximal histidine below the heme plane. Even though they bind oxygen (O2), the proposed functions of these proteins are linked to nitric oxide (NO) metabolism, redox balance and energy maintenance, especially in hypoxic conditions, to a higher extent than oxygen transport.

One aim of this thesis was to generate mutant(s) with altered characteristics using site-directed mutagenesis and investigate the role of these residues. One residue in particular was a conserved cysteine residue located at position 86 (C86A) in sugar beet (Beta vulgaris ssp. vulgaris) nsPgb BvPgb1.2. After crystallization and structure determination, this cysteine-to-alanine substitution had no impact on the tertiary and quaternary structure but major implications regarding functionality were observed. The mutant had faster autoxidation rate and increased thermal stability, while the wild-type (WT) protein showed higher peroxidase activity. Both species appeared as homodimers and did not show any heme loss, unlike human hemoglobin.

To gain more information regarding the intra- and intermolecular interactions in
nsPgbs, triple-labeled BvPgb1.2 was expressed, purified and analyzed using 2D
Nuclear Magnetic Resonance (NMR). 83% of the expected amide cross-peaks were assigned. The majority of the non-assigned residues were located in G and H αhelices, which are proposed to be an important area of dimer interactions. Hydrogen bonding between specific residues (T53 and E123) and a hydrophobic cluster in opposing monomers were suggested to be important in dimer formation. Structure predictions were conducted to gain knowledge regarding the role of the conserved cysteine in dimerization and mapping the lost residues in the crystal structure. These residues were located at highly flexible regions in the N- and C-termini, as well as in the loops for helices CD/DE. Small angle X-ray scattering (SAXS) confirmed maintained a dimeric quaternary structure at low protein concentrations (~0.15 mg/ml) and physiological salt concentrations for WT and C86A. Different proteindependent oligomerization tendencies were observed for the proteins, where the effects for C86A were most prominent. High degree of dimerization was observed for both proteins, not affected by the imposed cysteine-substitution to great extent.

To evaluate the biotechnical applicability of these proteins, encapsulation of
BvPgb1.2 and Mb were carried out using a lipid-based sponge phase system. High protein concentrations were correlated with increased aggregation tendencies, especially for BvPgb1.2. This effect seemed to be reversible upon agitation and the internal sponge phase structure was maintained. When analyzed with size exclusion chromatography (SEC), no protein leakage was detected for the nsPgb. To study toxicological effects of BvPgb1.2 WT, C86A and Mb, these proteins were labeled with fluorescent markers and studied in vivo using a zebrafish model. The globins were injected into brain and lateral muscle tissues to study potential accumulation and toxicity. Granules of C86A and Mb were detected in brain tissue, while all proteins were observed in the muscle tissues. In general, either no/low oxidative stress was observed for the penta- or hexacoordinated globins, except for the most concentrated granules of BvPgb1.2 WT, indicating good tolerance in this model system.

The results highlighted in this thesis provide valuable insights in order to
improve/strengthen several areas within biotechnology, including potential
generation of resilient crops, iron supplements and oxygen therapeutics.

Abstract (Swedish)

Phytoglobins (Pgb) (plant hemoglobins) are heme-containing proteins and less well known in comparison to the mammalian counterparts hemoglobin (Hb) and
myoglobin (Mb). They share the same secondary structure as other globin-related proteins, six to eight α-helices comprising the myoglobin-fold (Mb-fold) with one prosthetic heme moiety per subunit. Pgbs are divided into different classes, where the non-symbiotic Pgbs (nsPgb) are found in vast number of plants. Unlike their mammalian counterparts, nsPgbs usually have an additional coordination site interacting with the iron called the distal histidine, in addition to the proximal histidine below the heme plane. Even though they bind oxygen (O2), the proposed functions of these proteins are linked to nitric oxide (NO) metabolism, redox balance and energy maintenance, especially in hypoxic conditions, to a higher extent than oxygen transport.

One aim of this thesis was to generate mutant(s) with altered characteristics using site-directed mutagenesis and investigate the role of these residues. One residue in particular was a conserved cysteine residue located at position 86 (C86A) in sugar beet (Beta vulgaris ssp. vulgaris) nsPgb BvPgb1.2. After crystallization and structure determination, this cysteine-to-alanine substitution had no impact on the tertiary and quaternary structure but major implications regarding functionality were observed. The mutant had faster autoxidation rate and increased thermal stability, while the wild-type (WT) protein showed higher peroxidase activity. Both species appeared as homodimers and did not show any heme loss, unlike human hemoglobin.

To gain more information regarding the intra- and intermolecular interactions in
nsPgbs, triple-labeled BvPgb1.2 was expressed, purified and analyzed using 2D
Nuclear Magnetic Resonance (NMR). 83% of the expected amide cross-peaks were assigned. The majority of the non-assigned residues were located in G and H αhelices, which are proposed to be an important area of dimer interactions. Hydrogen bonding between specific residues (T53 and E123) and a hydrophobic cluster in opposing monomers were suggested to be important in dimer formation. Structure predictions were conducted to gain knowledge regarding the role of the conserved cysteine in dimerization and mapping the lost residues in the crystal structure. These residues were located at highly flexible regions in the N- and C-termini, as well as in the loops for helices CD/DE. Small angle X-ray scattering (SAXS) confirmed maintained a dimeric quaternary structure at low protein concentrations (~0.15 mg/ml) and physiological salt concentrations for WT and C86A. Different proteindependent oligomerization tendencies were observed for the proteins, where the effects for C86A were most prominent. High degree of dimerization was observed for both proteins, not affected by the imposed cysteine-substitution to great extent.

To evaluate the biotechnical applicability of these proteins, encapsulation of
BvPgb1.2 and Mb were carried out using a lipid-based sponge phase system. High protein concentrations were correlated with increased aggregation tendencies, especially for BvPgb1.2. This effect seemed to be reversible upon agitation and the internal sponge phase structure was maintained. When analyzed with size exclusion chromatography (SEC), no protein leakage was detected for the nsPgb. To study toxicological effects of BvPgb1.2 WT, C86A and Mb, these proteins were labeled with fluorescent markers and studied in vivo using a zebrafish model. The globins were injected into brain and lateral muscle tissues to study potential accumulation and toxicity. Granules of C86A and Mb were detected in brain tissue, while all proteins were observed in the muscle tissues. In general, either no/low oxidative stress was observed for the penta- or hexacoordinated globins, except for the most concentrated granules of BvPgb1.2 WT, indicating good tolerance in this model system.

The results highlighted in this thesis provide valuable insights in order to
improve/strengthen several areas within biotechnology, including potential
generation of resilient crops, iron supplements and oxygen therapeutics.

• author: Christensen, Simon
• supervisor: Leif Bülow; Mats Larsson; Johan Svensson Bonde
• opponent: Prof. Bäumler, Hans, Charité Universitätsmedizin, Germany.
• organization: Pure and Applied Biochemistry
• alternative title: Fytoglobiner: Utredning av Strukturella, Funktionella och Praktiska Särdrag för Icke-Symbiotiska Växt Hemoglobiner
• publishing date: 2023-04-14
• type: Thesis
• publication status: published
• subject: Biochemistry and Molecular Biology
• keywords: Hemoglobin, Fytoglobin, Hexakoordination, Karaktärisering, Protein design, Rekombinant protein production, Kromatografi, kristallografi, Dimerisering, Protein Dynamik
• pages: 94 pages
• defense location: Lecture Hall KC:B, Kemicentrum, Naturvetarvägen 14, Faculty of Engineering LTH, Lund University, Lund
• defense date: 2023-06-07 09:00:00
• ISBN: 978-91-7422-936-3
• ISBN: 978-91-7422-937-0
• language: English
• LU publication?: yes
• id: 235b9873-51b3-4c7b-839a-8270bc524b44
• date added to LUP: 2023-04-14 16:00:13
• date last changed: 2023-05-10 09:22:39