Biology subjects

Eichacker, L. A.

Publications and source records attributed to Eichacker, L. A..

4 recordsLinked to original sources

bioRxiv2026

Mass spectrometric identification of a PSI-LHCI-cytochrome b6f complex in both Arabidopsis stn7 and pph1

Native PAGE separation of digitonin-solubilized thylakoid stroma lamellae resolves PSI and cytochrome b6f into separate bands. Mass spectrometric analysis of the 1D native gel bands showed that the bands are baskets of co-migrating complexes along the molecular size axis. Hierarchical clustering confirmed the separation of dimeric cytochrome b6f from PSI in 1D LN-PAGE. Interval-zone free-flow electrophoresis separated the same extracts by charge in a flowing buffer film, and the continuous fractionation indicated co-migration of complexes without the in-gel structural hindrances. A second-dimension LN-PAGE separated the complexes co-migrating in iZE into complexes with defined molecular size, allowing in-gel mass spectrometry identification in the gel bands. Both genotypes showed a 720 kDa fluorescent band in which cytochrome f (PetA) and cytochrome b6 (PetB) join the PSI-LHCI node (stn7, d = 0.50; pph1, d = 0.34). Lhcb1 and Lhcb2 are absent from the 720 kDa node. The 753 kDa band is specific to pph1 (d = 0.38). Phosphorylated LHCII1,2 joins PSI-LHCI there, and PetB is detected in every slice of that band without becoming a leaf of the tree.

plant biology↗

bioRxiv2026

Interval-zone free-flow electrophoresis as a charge-specific dimension for native isolation of thylakoid membrane protein complexes

Native polyacrylamide gel electrophoresis is the standard first analytical step after detergent solubilization of thylakoid membranes. It separates photosystem supercomplexes by size and shape, but it forces a polydisperse mixture of protein-detergent particles through a gel of limited pore diameter. Interval-zone free-flow electrophoresis (iZE-FFE) performs that first electrophoretic step in free solution, without ampholytes or immobilines, and returns the complexes as a liquid fraction series ordered by net charge. We describe the operational cycle of iZE, show that bromophenol blue reports the charge density of {beta}-dodecylmaltoside micelles, and apply iZE to Arabidopsis thaliana thylakoid extracts from two sequential solubilization campaigns. After a two-step digitonin then digitonin/{beta}-dodecylmaltoside extraction (experiment 6874), SDS-PAGE of the iZE fractions is crowded: high-molecular mass protein-lipid assemblies of photosystem I and photosystem II release the same subunits into many wells. Native-PAGE of the same fractions resolves ATP-synthase from PSI, a free LHCII pool, anodic PSII, and cathodic PSI, including PSI with and without LHCII. A three-step extraction with a single pH working window (experiment 7154) confirms this charge order. Lowering residual detergent in consecutive extracts still produces partly solubilized high-mass assemblies, whereas a final {beta}-dodecylmaltoside step releases the C2S2M2/C2S2M/C2S2 PSII series and a single PSI band. iZE is therefore a charge-first, orthogonal dimension for native thylakoid biochemistry. Native-PAGE, not SDS-PAGE, is the proper second dimension while solubilization remains incomplete.

plant biology↗

bioRxiv2026

Chlorophyll binding to Cytochrome b6f precedes photosystem I and II in barley etioplasts

Chlorophyll (Chl) is essential for oxygenic photosynthesis, binding to membrane proteins for light harvesting and electron transfer. In angiosperms, Chl synthesis is halted in darkness, preventing accumulation of Chl-binding photosynthetic complexes in etioplasts. However, etioplasts assemble a dimeric Cytochrome b6f (Cyt b6f) complex, uniquely binding protochlorophyll (Pchl), the esterified derivative of protochlorophyllide (Pchlide). This indicates an evolutionarily conserved structural or functional role for Pchl distinct from the Chl bound in Cyt b6f in chloroplasts. Here we show that upon light-induced Chl synthesis in-vivo and in-vitro, Chl accumulation in Cyt b6f dimers precedes photosystems I and II. We find that chlorophyllide and Chl bind to the light-harvesting-like protein 3 (LIL3), supporting a role for LIL3 in early Chl allocation that extends its described role in stabilizing geranylgeranyl reductase. We determine a dissociation constant of 246.6 {+/-} 37 nM for Chlide binding to LIL3 in-vitro and show that Cyt b6f monomers and LIL3 co-migrate with Chlide in native PAGE, whereas Cyt b6f dimers and LIL3 co-migrate with Chl. These results indicate that Chlide binding to LIL3 chaperones esterification to Chl and reduction of geranylgeraniol, and that Chl release with Cyt b6f dimerization prioritizes Chl binding to Cyt b6f assembly during de-etiolation.

plant biology↗

bioRxiv2022

Observation of a single protein by ultrafast X-ray diffraction

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory1, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes2. This was first demonstrated on biological samples a decade ago on the giant mimivirus3. Since then a large collaboration4 has been pushing the limit of the smallest sample that can be imaged5,6. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter7 is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

biophysics↗