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Modulation of protein folding rate of GFP through beta-turn engineering
( Bharat Madan ),( Govindan Raghunathan ),( Sriram Sokalingam ),( Thangappan Jayaraman ),( Sungwan Park ),이선구 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1
Control of the protein folding rate is very important in various biological studies and biotechnology. The present study examined the possibility of modulating the protein folding rate through beta-turn engineering of green fluorescence protein (GFP). A type II’ two residue beta-turn in GFP was targeted to generate various sets of mutants. The design of mutants was based on the rationale that residues i+1 and i+2 of a beta-turn have defined residue preferences, and their perturbation affects the rate of protein folding. The in vitro refolding kinetics performed with purified variants showed the variantion of GFP folding rate as expected. This kind of study would be useful in the modulation of in vivo activities of recombinant proteins which have been generally controlled at transcription and translation levels.
Modulation of Intracellular Protein Activity at Level of Protein Folding by Beta-turn Engineering
Bharat Madan,이선구 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.3
Control of the intracellular protein activity is veryimportant in various biological studies and biotechnology. This has generally been achieved at the transcription andtranslation levels. Although control of the intracellularactivity at the protein folding level is conceptually possible,but there have been few studies. The present studyexamined this possibility by modulating the in vivo proteinfolding rate of green fluorescence protein (GFP) throughbeta-turn engineering. A type II’ two residue beta-turn inGFP was targeted to generate two sets of mutants. First, aswitch-off mutant was designed to stop the protein activitycompletely. The modulation mutants were then constructedto change the rates of GFP folding. The design of mutantswas based on the rationale that residues i+1 and i+2 of abeta-turn have defined residue preferences, and theirperturbation affects the rate of protein folding. The in vivofluorescence activity of the designed GFP variants wasswitched off and modulated as expected. The change in thein vivo folding patterns of the mutants was confirmed bySDS-PAGE and found to be similar to the intracellularfluorescence activities of the mutants. The in vitro refoldingkinetics performed with purified variants showed correlationswith the in vivo folding patterns. These results showed thatthe beta-turns in a protein can be a target for modulatingthe in vivo protein folding pattern and activity.
Sequence and Structural Features of Subsite Residues in GH10 and GH11 Xylanases
Bharat Madan,이선구 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.3
Xylanases are the enzymes that breakdown complex plant cell wall polysaccharide xylan into xylose by hydrolysing the β-(1→4) glycosidic linkage between xylosides. They mainly belong to the families GH10 and GH11 of the glycoside hydrolase class of enzymes. GH10 xylanases have (α/β)8-barrel type of fold whereas GH11 xylanases have β-jelly roll type of fold. Both enzymes have several substrate binding subsites. This study analysed in detail the sequence and structural conservation of subsites residues by examining their 3D structures crystallized with homoxylan or its non-hydrolysable form as substrate. A total of 19 structures from GH10 and 6 structures from GH11 were analysed. It was found that in GH10 the subsites -3 to -1 consisted of conserved residues, whereas in GH11 subsites -1, -3 and +1 were found to be conserved. The substrate and subsite interaction analysed based on the presence of h-bonds and CH-π interactions showed that Face-to-Face or Edge-to-Face CH-π interactions are formed in the subsites of GH10, whereas such specific CH-π interactions were no at all observed in case of GH11 xylanases. The spatial conservation of subsite residues was also analysed using a distance matrix based approach. It was found that in GH10 xylanases conserved residues have conserved spatial position of those residues as opposed to GH11 enzymes where in subsites -2 and +2 conserved residues showed non-conservation in their spatial positions. The results presented in this study can be used in discovering new xylanases and in the engineering highly efficient xylanases.
2P-364 Study on the features of subsites in xylanases
( Bharat Madan ),이선구 한국공업화학회 2017 한국공업화학회 연구논문 초록집 Vol.2017 No.1
The crystal structures for the GH10 and GH11 family xylanases revealed that both the families have different structural folds. The family 10 xylanases consists of (α/β)8-barrel type of fold. On the other hand, the family 11 xylanases shows a β-jelly roll type of fold consisting of antiparallel beta-sheets linked by loops and short helices which resembles the shape of a right hand. The substrate binding site that accommodate xylose moiety is known as a subsite. These subsites often contain residues such as tyrosine and tryptophan which can pack effectively against the xylose ring and other polar amino acids which can form hydrogen bonds with the substrate molecule. In this study, the sequence and structural features of the subsites in the xyalanases were chraterized by sequence and structural alignments. The substrate binding modes in the subsites were also computationally characterized.
Engineering a beta-turn in green fluorescent protein to a foreign loop
Bharat Madan,이선구,Jayaraman Thangappan 한국공업화학회 2016 Journal of Industrial and Engineering Chemistry Vol.33 No.-
A type I beta-turn in green fluorescent protein (GFP) was engineered to a foreign loop. Moleculardynamics simulation study showed that the addition of foreign loop into GFP did not have a negativeinfluence on the conformation stability of GFP structure, but the GFP variant with the foreign loopsequence was completely misfolded in real folding conditions. The co-incorporation of the enhancingmutations for GFP folding made it possible to generate a foldable and active GFP variant with the foreignloop sequence, although the folding efficiency and specific activity of the GFP were negatively affected bythe introduced loop.