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Protein science 32(2), e4567 (2023). doi:10.1002/pro.4567, DNA-binding proteins from starved cells (Dps) are small multifunctional nanocages expressed by prokaryotes in acute oxidative stress conditions or during the starvation-induced stationary phase, as a bacterial defense mechanism. Dps proteins protect bacterial DNA from damage by either direct binding or by removing precursors of reactive oxygen species from solution. The DNA-binding properties of most Dps proteins studied so far are related to their unordered, flexible, N- and C-terminal extensions. In a previous work, we revealed that the N-terminal tails of Deinoccocus grandis Dps shift from an extended to a compact conformation depending on the ionic strength of the buffer and detected a novel high-spin ferrous iron center in the proximal ends of those tails. In this work, we further explore the conformational dynamics of the protein by probing the effect of divalent metals binding to the tail by comparing the metal-binding properties of the wild-type protein with a binding site-impaired D34A variant using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism, and small-angle X-ray scattering. The N-terminal ferrous species was also characterized by Mössbauer spectroscopy. The results herein presented reveal that the conformation of the N-terminal tails is altered upon metal binding in a gradual, reversible, and specific manner. These observations may point towards the existence of a regulatory process for the DNA-binding properties of Dps proteins through metal binding to their N- and/or C-terminal extensions., Published by Protein Society, Bethesda, Md.

Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of messenger RNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochemical characteristics of the LNPs, such as particle size, morphology, and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after intravenous injection. Notably, a higher protein secretion with a reduced immunostimulatory response was observed when compared to systems based on polyethylene glycol (PEG) lipids. pSarcosinylated nanocarriers showed a lower proinflammatory cytokine secretion and reduced complement activation compared to PEGylated LNPs. In summary, the described pSar-based LNPs enable safe and potent delivery of mRNA, thus signifying an excellent basis for the development of PEG-free RNA therapeutics.

Lab on a chip 16(7), 1161 - 1170(2016). doi:10.1039/C5LC01580D, We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7–4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines., Published by RSC, Cambridge

Multifunctional enzyme type 2 (MFE-2) forms part of the fatty acid β-oxidation pathway in peroxisomes. MFE-2s from various species reveal proteins with structurally homologous functional domains assembled in different compilations. Crystal structures of all domain types are known. SAXS data from human, fruit fly and Caenorhabditis elegans MFE-2s and their constituent domains were collected, and both ab initio and rigid body models constructed. Location of the putative substrate binding helper domain SCP-2L (sterol carrier protein 2-like), which is not part of MFE-2 protein in every species and not seen as part of any previous MFE-2 structures, was determined. The obtained models of human and C. elegans MFE-2 lend a direct structural support to the idea of the biological role of SCP-2L.Structured summary of protein interactionsHsMFE-2 and HsMFE-2 bind by x ray scattering (View interaction)CeMFE-2 and CeMFE-2 bind by x ray scattering (View interaction)DmMFE-2 and DmMFE-2 bind by x ray scattering (View interaction)

Acta crystallographica / A 72(a1), s14 - s14(2016). doi:10.1107/S2053273316099770, Published by Blackwell, Oxford [u.a.]