Modern computational tools can predict the mutational effects on protein stability, sometimes at the expense of activity or solubility. Here, we investigate two homologous computationally stabilized haloalkane dehalogenases: (i) the soluble thermostable DhaA115 (Tmapp = 74 °C) and (ii) the poorly soluble and aggregating thermostable LinB116 (Tmapp = 65 °C), together with their respective wild-type variants. The intriguing difference in the solubility of these highly homologous proteins has remained unexplained for three decades. We combined experimental and in-silico techniques and examined the effects of stabilization on solubility and aggregation propensity. A detailed analysis of the unfolding mechanisms in the context of aggregation explained the negative consequences of stabilization observed in LinB116. With the aid of molecular dynamics simulations, we identified regions exposed during the unfolding of LinB116 that were later found to exhibit aggregation propensity. Our analysis identified cryptic aggregation-prone regions and increased surface hydrophobicity as key factors contributing to the reduced solubility of LinB116. This study reveals novel molecular mechanisms of unfolding for hyperstabilized dehalogenases and highlights the importance of contextual information in protein engineering to avoid the negative effects of stabilizing mutations on protein solubility.

• MeSH
• hydrofobní a hydrofilní interakce MeSH
• hydrolasy * chemie   metabolismus   genetika   MeSH
• proteinové agregáty * MeSH
• rozbalení proteinů * MeSH
• rozpustnost MeSH
• simulace molekulární dynamiky MeSH
• stabilita proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy * MeSH
• proteinové agregáty * MeSH

Fibroblast growth factors (FGFs) control organ morphogenesis during development as well as tissue homeostasis and repair in the adult organism. Despite their importance, many mechanisms that regulate FGF function are still poorly understood. Interestingly, the thermodynamic stability of 22 mammalian FGFs varies widely, with some FGFs remaining stable at body temperature for more than 24 h, while others lose their activity within minutes. How thermodynamic stability contributes to the function of FGFs during development remains unknown. Here we show that FGF10, an important limb and lung morphogen, exists as an intrinsically unstable protein that is prone to unfolding and is rapidly inactivated at 37 °C. Using rationally driven directed mutagenesis, we have developed several highly stable (STAB) FGF10 variants with a melting temperature of over 19 °C more than that of wildtype FGF10. In cellular assays in vitro, the FGF10-STABs did not differ from wildtype FGF10 in terms of binding to FGF receptors, activation of downstream FGF receptor signaling in cells, and induction of gene expression. In mouse embryonal lung explants, FGF10-STABs, but not wildtype FGF10, suppressed branching, resulting in increased alveolarization and expansion of epithelial tissue. Similarly, FGF10-STAB1, but not FGF10 wildtype, inhibited the growth of mouse embryonic tibias and markedly altered limb morphogenesis when implanted into chicken limb buds, collectively demonstrating that thermal instability should be considered an important regulator of FGF function that prevents ectopic signaling. Furthermore, we show enhanced differentiation of human iPSC-derived lung organoids and improved regeneration in ex vivo lung injury models mediated by FGF10-STABs, suggesting an application in cell therapy.

• Klíčová slova
• Development, FGF10, Fibroblast growth factor, Lung, Morphogen, Stability,
• MeSH
• fibroblastový růstový faktor 10 * metabolismus   genetika   chemie   MeSH
• lidé MeSH
• myši MeSH
• plíce metabolismus   embryologie   MeSH
• receptory fibroblastových růstových faktorů metabolismus   MeSH
• signální transdukce * MeSH
• stabilita proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Fgf10 protein, mouse MeSH Prohlížeč
• fibroblastový růstový faktor 10 * MeSH
• receptory fibroblastových růstových faktorů MeSH

Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.

• Klíčová slova
• Protein flexibility, Stabilized fibroblast growth factor 2, X-ray structural analysis,
• Publikační typ
• časopisecké články MeSH

FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.

• Klíčová slova
• Fibroblast growth factor 21, Protein engineering, Protein stabilization,
• Publikační typ
• časopisecké články MeSH

Thermostable proteins find their use in numerous biomedical and biotechnological applications. However, the computational design of stable proteins often results in single-point mutations with a limited effect on protein stability. However, the construction of stable multiple-point mutants can prove difficult due to the possibility of antagonistic effects between individual mutations. FireProt protocol enables the automated computational design of highly stable multiple-point mutants. FireProt 2.0 builds on top of the previously published FireProt web, retaining the original functionality and expanding it with several new stabilization strategies. FireProt 2.0 integrates the AlphaFold database and the homology modeling for structure prediction, enabling calculations starting from a sequence. Multiple-point designs are constructed using the Bron-Kerbosch algorithm minimizing the antagonistic effect between the individual mutations. Users can newly limit the FireProt calculation to a set of user-defined mutations, run a saturation mutagenesis of the whole protein or select rigidifying mutations based on B-factors. Evolution-based back-to-consensus strategy is complemented by ancestral sequence reconstruction. FireProt 2.0 is significantly faster and a reworked graphical user interface broadens the tool's availability even to users with older hardware. FireProt 2.0 is freely available at http://loschmidt.chemi.muni.cz/fireprotweb.

• Klíčová slova
• B-factor, ancestral, back-to-consensus, epistasis, evolution, force-field, multiple-point mutant, protein engineering, saturation mutagenesis, thermostability,
• MeSH
• algoritmy * MeSH
• internet MeSH
• mutace MeSH
• proteiny * genetika   chemie   MeSH
• stabilita proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH

The fibroblast growth factors (FGF) family holds significant potential for addressing chronic diseases. Specifically, recombinant FGF18 shows promise in treating osteoarthritis by stimulating cartilage formation. However, recent phase 2 clinical trial results of sprifermin (recombinant FGF18) indicate insufficient efficacy. Leveraging our expertise in rational protein engineering, we conducted a study to enhance the stability of FGF18. As a result, we obtained a stabilized variant called FGF18-E4, which exhibited improved stability with 16 °C higher melting temperature, resistance to trypsin and a 2.5-fold increase in production yields. Moreover, the FGF18-E4 maintained mitogenic activity after 1-week incubation at 37 °C and 1-day at 50 °C. Additionally, the inserted mutations did not affect its binding to the fibroblast growth factor receptors, making FGF18-E4 a promising candidate for advancing FGF-based osteoarthritis treatment.

• Klíčová slova
• Computer-assisted stabilization, FGF-18, Fibroblast growth factor, Improved yield, Protease, Resistance to, Thermostability,
• Publikační typ
• časopisecké články MeSH

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.

• Publikační typ
• časopisecké články MeSH

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.

• Klíčová slova
• PROSS, Protein expression, Protein stability, Recombinant proteins, Rosetta,
• MeSH
• algoritmy * MeSH
• dánio pruhované MeSH
• Escherichia coli metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• molekulární modely MeSH
• proteiny chemie   metabolismus   MeSH
• rozpustnost MeSH
• rychlé screeningové testy MeSH
• stabilita proteinů * MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH

Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T m = 73.5 °C; ΔT m > 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.

• Publikační typ
• časopisecké články MeSH

Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure-function relationships of >100.000 members of the α/β-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14-12.6 μM-1 s-1) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005-0.014 μM-1 s-1). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii-iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.

• Klíčová slova
• Enzyme kinetics, Fluorescent substrate, Haloalkane dehalogenase, Mechanism, Protein labeling,
• Publikační typ
• časopisecké články MeSH