- MeSH
- antioxidancia MeSH
- DNA MeSH
- rostlinné extrakty MeSH
- silymarin * MeSH
- Publikační typ
- dopisy MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.
- MeSH
- databáze proteinů MeSH
- interleukin-10 metabolismus MeSH
- konformace proteinů MeSH
- počítačová simulace MeSH
- proteinové inženýrství MeSH
- proteiny chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- ribozomy metabolismus MeSH
- řízená evoluce molekul metody MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) catalyze the cleavage of the glycosidic bond between the aglycone and the disaccharide rutinose (α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranose) of specific flavonoid glycosides such as rutin (quercetin 3-O-rutinoside). Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three-dimensional structure of a rutinosidase determined at 1.27-Å resolution. The rutinosidase from Aspergillus niger K2 (AnRut), a member of glycoside hydrolase family GH-5, subfamily 23, was heterologously produced in Pichia pastoris. The X-ray structure of AnRut is represented by a distorted (β/α)8 barrel fold with its closest structural homologue being an exo-β-(1,3)-glucanase from Candida albicans (CaExg). The catalytic site is located in a deep pocket with a striking structural similarity to CaExg. However, the entrance to the active site of AnRut has been found to be different from that of CaExg - a mostly unstructured section of ~ 40 residues present in CaExg is missing in AnRut, whereas an additional loop of 13 amino acids partially covers the active site of AnRut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of AnRut. Unexpectedly, quercetin 3-O-glucoside was found to be a better substrate than rutin, and thus, AnRut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with in silico modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of AnRut has been revised. DATABASES: The nucleotide sequence of the rutinosidase-encoding gene is available in the GenBank database under the accession number MN393234. Structural data are available in the PDB database under the accession number 6I1A. ENZYME: α-l-Rhamnosyl-β-d-glucosidase (EC 3.2.1.168).
- MeSH
- Aspergillus niger enzymologie MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- glykosidhydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- mutace MeSH
- oxidace-redukce MeSH
- rutin chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Most biological processes involve water, and the interactions of biomolecules with water affect their structure, function and dynamics. SCOPE OF REVIEW: This review summarizes the current knowledge of protein and nucleic acid interactions with water, with a special focus on the biomolecular hydration layer. Recent developments in both experimental and computational methods that can be applied to the study of hydration structure and dynamics are reviewed, including software tools for the prediction and characterization of hydration layer properties. MAJOR CONCLUSIONS: In the last decade, important advances have been made in our understanding of the factors that determine how biomolecules and their aqueous environment influence each other. Both experimental and computational methods contributed to the gradually emerging consensus picture of biomolecular hydration. GENERAL SIGNIFICANCE: An improved knowledge of the structural and thermodynamic properties of the hydration layer will enable a detailed understanding of the various biological processes in which it is involved, with implications for a wide range of applications, including protein-structure prediction and structure-based drug design.
- MeSH
- nukleové kyseliny metabolismus MeSH
- proteiny metabolismus MeSH
- simulace molekulární dynamiky MeSH
- voda metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. The results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon-donor hydrogen bonds, OH-π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.
- MeSH
- aminokyseliny analýza MeSH
- databáze proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- proteiny chemie MeSH
- sekundární struktura proteinů MeSH
- voda analýza MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.
- MeSH
- interferon gama chemie metabolismus MeSH
- lidé MeSH
- missense mutace MeSH
- molekulární modely * MeSH
- receptory interferonů chemie genetika metabolismus MeSH
- sbalování proteinů * MeSH
- substituce aminokyselin * MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor.
- MeSH
- interferon gama chemie genetika metabolismus MeSH
- lidé MeSH
- povrchová plasmonová rezonance MeSH
- receptory interferonů chemie genetika metabolismus MeSH
- sbalování proteinů * MeSH
- simulace molekulární dynamiky * MeSH
- substituce aminokyselin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein structures contain highly complex systems of voids, making up specific features such as surface clefts or grooves, pockets, protrusions, cavities, pores or channels, and tunnels. Many of them are essential for the migration of solvents, ions and small molecules through proteins, and their binding to the functional sites. Analysis of these structural features is very important for understanding of structure-function relationships, for the design of potential inhibitors or proteins with improved functional properties. Here we critically review existing software tools specialized in rapid identification, visualization, analysis and design of protein tunnels and channels. The strengths and weaknesses of individual tools are reported together with examples of their applications for the analysis and engineering of various biological systems. This review can assist users with selecting a proper software tool for study of their biological problem as well as highlighting possible avenues for further development of existing tools. Development of novel descriptors representing not only geometry, but also electrostatics, hydrophobicity or dynamics, is needed for reliable identification of biologically relevant tunnels and channels.
Počítačová předpověď proteinových mutací vedoucí ke zvýšení jejich afinity k cílovému proteinu není běžně používaný, ale užitečný nástroj proteinového inženýrství. Zde ukazujeme, že dostupnými výpočetními prostředky je možné předpovědět zvýšení afinity receptoru 1 k interferonu-γ. U dvou, ze čtrnácti předpovězených mutant, došlo k pětinásobnému zvýšení disociační rovnovážné konstanty Kd jak bylo ukázáno měřením povrchovou plasmonovou resonancí, SPR.
Computer design of mutations increasing affinity to the target protein is not a standard but useful tool of protein engineering. Here we show that it is possible to apply relatively simple and accessible computer techniques based on empirical potential to predict mutants of receptor 1 to interferon-γ. Two out of fourteen designed mutants showed about five-fold increase of dissociation equilibrium constant K d as measured by surface plasmon resonance, SPR.
Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K(d) values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- genová knihovna MeSH
- interferon gama metabolismus MeSH
- katalytická doména MeSH
- lidé MeSH
- molekulární modely MeSH
- mutace MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- sérový albumin metabolismus MeSH
- Streptococcus chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
