scholarly journals Enhanced Thermostability of D-Psicose 3-Epimerase from Clostridium bolteae through Rational Design and Engineering of New Disulfide Bridges

2021 ◽  
Vol 22 (18) ◽  
pp. 10007
Author(s):  
Jingyi Zhao ◽  
Jing Chen ◽  
Huiyi Wang ◽  
Yan Guo ◽  
Kai Li ◽  
...  
Keyword(s):  
Industrial Application ◽  
Rational Design ◽  
Spatial Configuration ◽  
Disulfide Bridge ◽  
Dynamics Simulation ◽  
Disulfide Bridges ◽  
Unfolded Protein ◽  
Bond Network ◽  
Low Calorie Sweetener ◽  
Clostridium Bolteae

D-psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to D-psicose (aka D-allulose, a low-calorie sweetener), but its industrial application has been restricted by the poor thermostability of the naturally available enzymes. Computational rational design of disulfide bridges was used to select potential sites in the protein structure of DPEase from Clostridium bolteae to engineer new disulfide bridges. Three mutants were engineered successfully with new disulfide bridges in different locations, increasing their optimum catalytic temperature from 55 to 65 °C, greatly improving their thermal stability and extending their half-lives (t1/2) at 55 °C from 0.37 h to 4–4.5 h, thereby greatly enhancing their potential for industrial application. Molecular dynamics simulation and spatial configuration analysis revealed that introduction of a disulfide bridge modified the protein hydrogen–bond network, rigidified both the local and overall structures of the mutants and decreased the entropy of unfolded protein, thereby enhancing the thermostability of DPEase.

scholarly journals In SilicoRational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

2013 ◽  
Vol 80 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Long Liu ◽  
Zhuangmei Deng ◽  
Haiquan Yang ◽  
Jianghua Li ◽  
Hyun-dong Shin ◽  
...  
Keyword(s):  
Systems Engineering ◽  
Rational Design ◽  
Catalytic Domain ◽  
Disulfide Bridge ◽  
Biochemical Properties ◽  
Bridge Engineering ◽  
High Catalytic Activity ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Triple Mutant

ABSTRACTHigh thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase fromAlkalimonas amylolyticathroughin silicorational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (kcat/Km) increased from 1.8 × 104to 2.4 × 104liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

Rational Design and Engineering of a Mutant Variant of Urate Oxidase as a Therapeutic Enzyme: A Molecular Dynamics Simulation Approach

2017 ◽  
Vol 13 (1) ◽  
pp. 30-38
Author(s):  
Sajjad Ahrari ◽  
Fatemeh Dabbagh ◽  
Sobhan Ahrari ◽  
Younes Ghasemi ◽  
Navid Mogharrab ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Rational Design ◽  
Dynamics Simulation ◽  
Urate Oxidase ◽  
Simulation Approach ◽  
Mutant Variant

The effect of amino substituents on the interactions of quinazolone derivatives with c-KIT G-quadruplex: insight from molecular dynamics simulation study for rational design of ligands

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76642-76650 ◽  
Author(s):  
Kiana Gholamjani Moghaddam ◽  
Seyed Majid Hashemianzadeh
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Simulation Study ◽  
Rational Design ◽  
Ligand Design ◽  
Dynamics Simulation ◽  
Rational Ligand Design ◽  
G Quadruplex ◽  
Ligand Interactions ◽  
Insight Into

Our study provides insight into the effect of different substituents on the G-quadruplex–ligand interactions which helps us rational ligand design.

scholarly journals Rational design of new NO and redox sensitivity into connexin26 hemichannels

Open Biology ◽  
2015 ◽  
Vol 5 (2) ◽  
pp. 140208 ◽  
Author(s):  
Louise Meigh ◽  
Daniel Cook ◽  
Jie Zhang ◽  
Nicholas Dale
Keyword(s):  
Redox Potential ◽  
Rational Design ◽  
Salt Bridge ◽  
Disulfide Bridge ◽  
Wild Type ◽  
No Donors ◽  
Bridge Formation ◽  
Redox Sensitivity ◽  
Intracellular Redox

CO 2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanisms. The mutation K125C gives potential for nitrosylation on Cys125 and formation of an SNO bridge to R104 of the neighbouring subunit. Unlike wild-type Cx26 hemichannels, which are insensitive to NO and NO 2 − , hemichannels comprising Cx26 K125C can be opened by NO 2 − and NO donors. However, NO 2 − was unable to modulate the doubly mutated (K125C, R104A) hemichannels, indicating that an inter-subunit bridge between C125 and R104 is required for the opening action of NO 2 − . In a further test, we introduced two mutations into Cx26, K125C and R104C, to allow disulfide bridge formation across the inter-subunit boundary. These doubly mutated hemichannels open in response to changes in intracellular redox potential.

scholarly journals Plant purple acid phosphatases - genes, structures and biological function.

2003 ◽  
Vol 50 (4) ◽  
pp. 1245-1256 ◽  
Author(s):  
Mariusz Olczak ◽  
Bronisława Morawiecka ◽  
Wiesław Watorek
Keyword(s):  
Disulfide Bridge ◽  
Acid Phosphatases ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Inorganic Pyrophosphate ◽  
Manganese Ion ◽  
Substrate Preferences ◽  
Purple Acid Phosphatases ◽  
Single Polypeptide ◽  
Monomeric Proteins

The properties of plant purple acid phosphatases (PAPs), metallophosphoesterases present in some bacteria, plants and animals are reviewed. All members of this group contain a characteristic set of seven amino-acid residues involved in metal ligation. Animal PAPs contain a binuclear metallic center composed of two irons, whereas in plant PAPs one iron ion is joined by zinc or manganese ion. Among plant PAPs two groups can be distinguished: small PAPs, monomeric proteins with molecular mass around 35 kDa, structurally close to mammalian PAPs, and large PAPs, homodimeric proteins with a single polypeptide of about 55 kDa. Large plant PAPs exhibit two types of structural organization. One type comprises enzymes with subunits bound by a disulfide bridge formed by cysteines located in the C-terminal region around position 350. In the second type no cysteines are located in this position and no disulfide bridges are formed between subunits. Differences in structural organisation are reflected in substrate preferences. Recent data reveal in plants the occurrence of metallophosphoesterases structurally different from small or large PAPs but with metal-ligating sequences characteristic for PAPs and expressing pronounced specificity towards phytate or diphosphate nucleosides and inorganic pyrophosphate.

scholarly journals Sindbis Virus Glycoprotein E1 Is Divided into Two Discrete Domains at Amino Acid 129 by Disulfide Bridge Connections

2000 ◽  
Vol 74 (19) ◽  
pp. 9313-9316 ◽  
Author(s):  
Brett S. Phinney ◽  
Dennis T. Brown
Keyword(s):  
Amino Acid ◽  
Disulfide Bonds ◽  
Sindbis Virus ◽  
Disulfide Bridge ◽  
Membrane Glycoprotein ◽  
Disulfide Bridges ◽  
Functional Regions ◽  
E1 Protein ◽  
Disulfide Linkages ◽  
Discrete Domains

ABSTRACT The E1 membrane glycoprotein of Sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (B. A. Abell and D. T. Brown, J. Virol. 67:5496–5501, 1993; R. P. Anthony, A. M. Paredes, and D. T. Brown, Virology 190:330–336, 1992). We have examined the disulfide bonds in E1 and have determined that the E1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the protein into two domains at amino acid 129. These separate sets of disulfides may stabilize and define the structural and functional regions of the E1 protein.

scholarly journals Molecular Dynamics Simulation and Kinetic Study of Fluoride Binding to V21C/V66C Myoglobin with a Cytoglobin-like Disulfide Bond

2020 ◽  
Vol 21 (7) ◽  
pp. 2512
Author(s):  
Lu-Lu Yin ◽  
Jia-Kun Xu ◽  
Xiao-Juan Wang ◽  
Shu-Qin Gao ◽  
Ying-Wu Lin
Keyword(s):  
Molecular Dynamics ◽  
Disulfide Bond ◽  
Rational Design ◽  
Double Mutant ◽  
Md Simulation ◽  
Experimental Studies ◽  
Heme Iron ◽  
Dynamics Simulation ◽  
Fluoride Ion ◽  
Artificial Proteins

Protein design is able to create artificial proteins with advanced functions, and computer simulation plays a key role in guiding the rational design. In the absence of structural evidence for cytoglobin (Cgb) with an intramolecular disulfide bond, we recently designed a de novo disulfide bond in myoglobin (Mb) based on structural alignment (i.e., V21C/V66C Mb double mutant). To provide deep insight into the regulation role of the Cys21-Cys66 disulfide bond, we herein perform molecular dynamics (MD) simulation of the fluoride–protein complex by using a fluoride ion as a probe, which reveals detailed interactions of the fluoride ion in the heme distal pocket, involving both the distal His64 and water molecules. Moreover, we determined the kinetic parameters of fluoride binding to the double mutant. The results agree with the MD simulation and show that the formation of the Cys21-Cys66 disulfide bond facilitates both fluoride binding to and dissociating from the heme iron. Therefore, the combination of theoretical and experimental studies provides valuable information for understanding the structure and function of heme proteins, as regulated by a disulfide bond. This study is thus able to guide the rational design of artificial proteins with tunable functions in the future.

Determination with Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight Mass Spectrometry of the Extensive Disulfide Bonding in Tarantula Venom Peptide Psalmopeotoxin I

2009 ◽  
Vol 15 (4) ◽  
pp. 517-529 ◽  
Author(s):  
Audrey Combes ◽  
Soo Jin Choi ◽  
Cyril Pimentel ◽  
Hervé Darbon ◽  
Dietmar Waidelich ◽  
...  
Keyword(s):  
Laser Desorption ◽  
Disulfide Bridge ◽  
Fragmentation Pattern ◽  
Laser Desorption Ionization ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Desorption Ionization ◽  
Native Peptides ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Psalmopeotoxin I (PcFK1) is a 33-residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. This peptide specifically inhibits the intra-erythrocyte stage of Plasmodium falciparum in vitro. It contains six cysteine residues forming three disulfide bridges and belongs to the superfamily of natural peptides containing the inhibitor cystine knot (ICK) fold. We produced the wild-type and mutated forms of the recombinant peptide to examine the mechanism of action of PcFK1. The purified toxins were consistently produced as two isobaric peptides (r-PcFK1-1 and r-PcFK1-2) with different retention properties but identical anti-plasmodial biological activity. Comparison of 15N-NMR heteronuclear single quantum correlation spectra revealed that although rPcFK1-1 was highly structured, rPcFK1-2 does not have a stable three-dimensional structure. We used high-energy collision-induced fragmentation of the peptides with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer to further investigate the structure of the native peptides in its natural form and produced in E. coli. The fragmentation spectra of the native peptides were very complex due to the occurrence in the spectrum of ions resulting from (1) cross-linking of fragments through a disulfide bridge and (2) asymmetric fragmentations of the disulfide bridges and (3) multiple neutral losses. The tandem mass spectrometry fragmentation pattern of r-PcFK1-1 was similar to that of the natural peptide isolated from crude venom, but r-PcFK1-2 had a clearly distinct fragmentation pattern, more closely resembling the fragmentation spectra of reduced and alkylated peptides. Observed ions could be attributed to specific fragments by comparing spectra between the wild-type and selected variants with point mutations (Y11W, R20T, Y26W, K28V). The disulfide connections in r-PcFK1-2 differed from those of the native peptide and showed a rare disulfide bridge between vicinal cysteine residues. The r-PcFK1_(R20T) variant showed a very limited fragmentation pattern when analyzed in positive mode but displayed much more fragmentation in negative mode pointing out the importance of the R20 residue in the fragmentation of PcFK1. Using the reductive matrix 1,5-diaminonaphtalene promoted strongly in source decay fragmentation of the peptides in MS mode. Our findings illustrated the critical role of the electronic environment around the central Cys18–Cys19 doublet in PcFK1 in internal fragmentation of the peptide.

Discovery of vascular endothelial growth factor receptor tyrosine kinase inhibitors by quantitative structure–activity relationships, molecular dynamics simulation and free energy calculation

RSC Advances ◽  
2016 ◽  
Vol 6 (42) ◽  
pp. 35402-35415 ◽  
Author(s):  
Juan Wang ◽  
Mao Shu ◽  
Xiaorong Wen ◽  
Yuanliang Wang ◽  
Yuanqiang Wang ◽  
...  
Keyword(s):  
Rational Design ◽  
Kinase Inhibitors ◽  
Growth Factor Receptor ◽  
Dynamics Simulation ◽  
Energy Calculation ◽  
Vascular Endothelial ◽  
Structure Activity ◽  
Quantitative Structure Activity Relationships ◽  
Combined Strategy ◽  
Endothelial Growth Factor

Employing the combined strategy to understand the features of KDR–ligands complexes, and provide a basis for rational design of inhibitors.

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