From a Droplet to a Fibril and from a Fibril to a Droplet: Intertwined Transition Pathways in Highly Dynamic Enzyme-Modulated Peptide-Adenosine Triphosphate Systems.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC1-13Kn, consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC1-13) merged with oligolysine segments of various lengths (Kn, n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC1-13Kn-digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered Kn segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC1-13Kn-ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Enabling High Activation of Glucose-6-Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression.

Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

H2 production under stress: [FeFe]­hydrogenases reveal strong stability in high pressure environments.

Hydrogenases are a diverse group of metalloenzymes that catalyze the conversion of H2 into protons and electrons and the reverse reaction. A subgroup is formed by the [FeFe]­hydrogenases, which are the most efficient enzymes of microbes for catalytic H2 conversion. We have determined the stability and activity of two [FeFe]­hydrogenases under high temperature and pressure conditions employing FTIR spectroscopy and the high-pressure stopped-flow methodology in combination with fast UV/Vis detection. Our data show high temperature stability and an increase in activity up to the unfolding temperatures of the enzymes. Remarkably, both enzymes reveal a very high pressure stability of their structure, even up to pressures of several kbars. Their high pressure-stability enables high enzymatic activity up to 2 kbar, which largely exceeds the pressure limit encountered by organisms in the deep sea and sub-seafloor on Earth.

Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase.

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

Residual Membrane Fluidity in Mycobacterial Cell Envelope Layers under Extreme Conditions Underlines Membrane-Centric Adaptation.

One of the routes for adaptation to extreme environments is via remodeling of cell membrane structure, composition, and biophysical properties rendering a functional membrane. Collective studies suggest some form of membrane feedback in mycobacterial species that harbor complex lipids within the outer and inner cell wall layers. Here, we study the homeostatic membrane landscape of mycobacteria in response to high hydrostatic pressure and temperature triggers using high pressure fluorescence, mass and infrared spectroscopies, NMR, SAXS, and molecular dynamics simulations. Our findings reveal that mycobacterial membrane possesses unique and lipid-specific pressure-induced signatures that attenuate progression to highly ordered phases. Both inner and outer membrane layers exhibit phase coexistence of nearly identical lipid phases keeping residual fluidity over a wide range of temperature and pressure, but with different sensitivities. Lipidomic analysis of bacteria grown under pressure revealed lipidome remodeling in terms of chain length, unsaturation, and specific long-chained characteristic mycobacterial lipids, rendering a fluid bacterial membrane. These findings could help understand how bacteria may adapt to a broad spectrum of harsh environments by modulating their lipidome to select lipids that enable the maintenance of a fluid functional cell envelope.