Stephan Hobe

High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization

We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, but may even promote counterion-independent self-assembly which in turn leads to silica precipitation. Enzymatic phosphorylation, a common modification in native silica biomineralizing peptides, can be used to modify the precipitation activity.

Secondary structure and dynamics study of the intrinsically disordered silica-mineralizing peptide P5S3 during silicic acid condensation and silica decondensation

The silica forming repeat R5 of sil1 from Cylindrotheca fusiformis was the blueprint for the design of P5S3, a 50‐residue peptide which can be produced in large amounts by recombinant bacterial expression. It contains 5 protein kinase A target sites and is highly cationic due to 10 lysine and 10 arginine residues. In the presence of supersaturated orthosilicic acid P5S3 enhances silica‐formation whereas it retards the dissolution of amorphous silica (SiO2) at globally undersaturated concentrations. The secondary structure of P5S3 during these 2 processes was studied by circular dichroism (CD) spectroscopy, complemented by nuclear magnetic resonance (NMR) spectroscopy of the peptide in the absence of silicate. The NMR studies of dual‐labeled (13C, 15N) P5S3 revealed a disordered structure at pH 2.8 and 4.5. Within the pH range of 4.5‐9.5 in the absence of silicic acid, the CD data showed a disordered structure with the suggestion of some polyproline II character. Upon silicic acid polymerization and during dissolution of preformed silica, the CD spectrum of P5S3 indicated partial transition into an α‐helical conformation which was transient during silica‐dissolution. The secondary structural changes observed for P5S3 correlate with the presence of oligomeric/polymeric silicic acid, presumably due to P5S3‐silica interactions. These P5S3‐silica interactions appear, at least in part, ionic in nature since negatively charged dodecylsulfate caused similar perturbations to the P5S3 CD spectrum as observed with silica, while uncharged ß‐d‐dodecyl maltoside did not affect the CD spectrum of P5S3. Thus, with an associated increase in α‐helical character, P5S3 influences both the condensation of silicic acid into silica and its decondensation back to silicic acid.

Spectroscopic Characterization of Reconstituted LHCII Which Contains Mainly CHL B and Xanthophylls.

In [1] the structure of the light-harvesting chlorophyll alb-protein complex, LHCII, was reported at 3.4 A resolution. The trimeric complex revealed twelve chlorophylls (Chls) per monomeric subunit, seven of which were in Van der Waals contact with two central xanthophyll molecules that were supposed to be luteins. The identities of the Chis (Chi a or Chl b) were not clear from the structure but the seven interior Chls next to the xanthophylls were assigned as Chl a. The main argument for this assignment was the following: excitations on Chi can lead to triplet formation on a ns time scale. The xanthophylls provide LHCII with the possibility of actively quenching Chl triplet states if the Chls and xanthophylls are in Van der Waals contact. If they were left unchecked, these triplet states would combine with oxygen molecules to create highly reactive and toxic singlet species. Since excitations on Chi b are being transferred to Chi a on a (sub)picosecond time scale, mainly Chi a triplets need to be quenched. This assignment was of course tentative. However, there was also some experimental support for it. Optically detected magnetic resonance (ODMR) experiments showed that triplets located on the xanthophylls did significantly influence the absorption spectrum of Chl a whereas virtually no effect on the Chl b absorption could be detected [2]. Later this effect was confirmed with the use of laser flash-induced triplet-minus-singlet spectroscopy [3, 4]. Although only contacts between xanthophyll and Chl a could be demonstrated in this way, it could not be ruled out that there might also be some contacts between xanthophyll and Chl b leaving the absorption of Chl b unaffected in the presence of xanthophyll triplets.