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KMID : 1094720130180020342
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Biotechnology and Bioprocess Engineering
2013 Volume.18 No. 2 p.342 ~ p.349
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A comparative study on the stability and structure of two different green fluorescent proteins in organic co-solvent systems
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Raghunathan Govindan
Sokalingam Sriram
Soundrarajan Nagasundarapandian
Munussami Ganapathiraman
Madan Bharat
Lee Sun-Gu
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Abstract
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Green fluorescent protein (GFP) has been used as a reporter marker in a wide range of biological and bioengineering studies. The expanded use of GFP in the field of biosensors, biochips and bio-conjugations requires the stability of GFP in organic co-solvent systems. This prompted us to examine the kinetic stability of two different GFP sequences, n-GFP and s-GFP, showing different folding robustness and thermodynamic stability, under a range of organic co-solvent systems. n-GFP and s-GFP are variants whose biophysical properties are comparable to wild type and super folder GFPs, respectively. The stability of n-GFP and s-GFP in 50% water-miscible organic solvents showed that s-GFP with higher thermodynamic stability exhibited much higher stability against organic solvents than n-GFP, which has lower thermodynamic stability. s-GFP was quite stable even in 90% organic solvents. Circular dichroism analysis confirmed that s-GFP maintained its native structure in organic co-solvent systems, whereas n-GFP showed structural variations under these conditions. Four highly fluctuating loop regions were identified from molecular dynamic simulations under the organic cosolvent conditions. A structural comparison of n-GFP and s-GFP suggested that the improved kinetic stability of s-GFP was due to its larger number of hydrogen bonds and salt-bridges that were present in four loop regions. This study suggests that thermodynamically stable s-GFP can be a good choice for use under harsh organic co-solvent conditions.
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KEYWORD
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green fluorescent protein, organic co-solvent system, stability to organic solvent, structural analysis, molecular dynamic simulation
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