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Koksch, B.

Publications and source records attributed to Koksch, B..

7 recordsLinked to original sources

Glycosaminoglycans Promote Amyloid-β Aggregation via Multivalent, pH-Dependent Interactions

Glycosaminoglycans (GAGs) are polyanionic polysaccharides that co-localize with amyloid-{beta} (A{beta}) deposits in Alzheimers disease, yet their mechanistic contribution to A{beta} aggregation remains unclear. Here, we show that GAGs function as pH-responsive electrostatic scaffolds that selectively accelerate A{beta}(1-42) aggregation under mildly acidic, endosomal conditions but not at neutral extracellular pH. Combining experimental and computational approaches, we identify protonated N-terminal histidines as key determinants of GAG binding. Weak interactions between GAGs and the charged Nterminal region of A{beta} promote conformational rearrangements that bring peptides into proximity and expose adjacent hydrophobic aggregation-prone segments, thereby facilitating peptide clustering. Kinetic analyses reveal that aggregation is enhanced in a way consistent with an apparent increase in effective peptide concentration, accelerating nucleation without altering the dominant aggregation pathway. Systematic variation of GAG chain length and sulfation level further demonstrates that aggregation enhancement requires a threshold degree of multivalency, consistent with a clustering-driven mechanism. Together, these findings establish a framework in which pH-dependent electrostatic interactions with GAGs act as molecular triggers of amyloid nucleation, providing insight into how cellular microenvironments regulate the earliest stages of Alzheimers disease pathology.

biophysics↗

HMGB1 B-box domain self-complexes promote protein-polyelectrolyte interactions.

HMGB1, a nuclear DNA-binding protein, can be secreted by activated immune cells or passively released from damaged cells. In such cases, HMGB1 functions as an alarmin that activates the immune system. Excessive inflammation may lead to pathogenesis, whereas this response can be dampened by polyanion binding, which impedes further receptor recognition. Moreover, HMGB1 is known to form liquid droplets in the cellular environment--a phase separation directly linked to its proper function. While the A-Box domain is believed to be primarily responsible for heparin binding due to its conserved binding site, the association and phase separation behavior of HMGB1 may be mediated by the B-box domain, owing to its extended hydrophobic regions. In this study, we first demonstrated that the B-box protein forms 30-nm large self-associates while maintaining its structure. Next, using molecularly sensitive EPR spectroscopy, we showed that the presence of these protein associations significantly enhances interactions with heparin. Notably, the local conformational changes induced by heparin are similar in both individual protein chains and their self-associated forms. To explain this effect, AlphaFold modeling was employed, revealing that the formation of protein multimers induces charge redistribution, resulting in an extended positively charged region that enhances electrostatic attraction to negatively charged polyanions, such as heparin.

biochemistry↗

Escherichia coli adapts metabolically to 6- and 7-fluoroindole, enabling proteome-wide fluorotryptophan substitution

Nature has scarcely evolved a biochemistry around fluorine. However, modern science proved fluorinated organic molecules to be suitable building blocks for biopolymers, from peptides and proteins up to entire organisms. Here, we conducted adaptive laboratory evolution (ALE) experiments to introduce fluorine into living microorganisms. By cultivating Escherichia coli with fluorinated indole analogues, we successfully evolved microbial cells capable of utilizing either 6-fluoroindole or 7-fluoroindole for growth. Our improved ALE protocols enabled us to overcome previous challenges and achieve consistent and complete adaptation of microbial populations to these unnatural molecules. In the ALE experiments, we supplied fluoroindoles to auxotrophic E. coli bacteria, exerting strong selective pressure that led to microbial adaptation and growth on monofluorinated indoles. Within the cells, these indoles were converted into corresponding amino acids (6- and 7-fluorotryptophan) and incorporated into the proteome at tryptophan sites. This study is a first step and establishes a strong foundation for further exploration of the mechanisms underlying fluorine-based life and how a formerly stressor (fluorinated indole) becomes a vital nutrient. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/559291v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1a3b17org.highwire.dtl.DTLVardef@1d716aorg.highwire.dtl.DTLVardef@10126d5org.highwire.dtl.DTLVardef@9c8a43_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Is the neuropeptide PEN a ligand of GPR83?

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second-messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

pharmacology and toxicology↗

Water network in the binding pocket of fluorinated BPTI-Trypsin complexes - insights from simulation and experiment

Structural waters in the S1 binding pocket of {beta}-trypsin are critical for the stabilization of the complex of {beta}-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by -aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly) and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific non-covalent interactions to the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly-BPTI-{beta}-trypsin complex (pdb: 7PH1). Both, experimental and computational data, show a step-wise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regards to energetic properties that could explain the increase in inhibitor strength. The analysis of the enthalpy and the entropy of the water molecules in the S1 binding pocket using Grid Inhomogeneous Solvation Theory confirms this result. Overall, fluorination systematically changes the binding affinity but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly. TOC GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/496563v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1177443org.highwire.dtl.DTLVardef@d20760org.highwire.dtl.DTLVardef@e252ecorg.highwire.dtl.DTLVardef@b22121_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Laboratory evolution of Escherichia coli enables life based on fluorinated amino acids

Organofluorine compounds are toxic to various living beings in different habitats. On the other hand, fluorine incorporation into single proteins via related amino acid analogues has become common practice in protein engineering. Thus, an essential question remains: can fluorinated amino acids generally be used as xeno-nutrients to build up biomass, or do large amounts of fluorine in the cells render them nonviable? To gain information about the effect of long-term exposure of a cellular proteome to fluorinated organic compounds, we constructed an experiment based on bacterial adaptation in artificial fluorinated habitats. We propagated Escherichia coli (E. coli) in the presence of either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of tryptophan (Trp) analogues. We found that full adaptation requires astonishingly few genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity and quality control of protein folding. These findings highlight the cellular mechanisms of the evolutionary adaption process to unnatural amino acids and provide the molecular foundation for novel and innovative bioengineering of microbial strains with potential for biotechnological applications.\n\nOne Sentence SummaryLaboratory evolution enabled for the first time Escherichia coli to use fluorinated indoles as essential precursors for protein synthesis by introducing few genetic mutations but large rearrangements in regulatory networks, membrane integrity and quality control of protein folding.

synthetic biology↗

Structural Determinants of Coiled Coil Mechanics

The natural abundance of coiled coil (CC) motifs in the cytoskeleton and the extracellular matrix suggests that CCs play a crucial role in the bidirectional mechanobiochemical signaling between cells and the matrix. Their functional importance and structural simplicity has allowed the development of numerous applications, such as protein-origami structures, drug delivery systems and biomaterials. With the goal of establishing CCs as nanomechanical building blocks, we investigated the importance of helix propensity and hydrophobic core packing on the mechanical stability of 4-heptad CC heterodimers. Using single-molecule force spectroscopy, we show that both parameters determine the force-induced dissociation in shear loading geometry; however, with different effects on the energy landscape. Decreasing the helix propensity lowers the transition barrier height, leading to a concomitant decrease in the distance to the transition state. In contrast, a less tightly packed hydrophobic core increases the distance to the transition state. We propose that this sequence-structure-mechanics relationship is evolutionarily optimized in natural CCs and can be used for tuning their mechanical properties in applications.

biophysics↗