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Characterization on the aggregation of self-aggregating green fluorescent protein variant
Raghunathan, G.,Munussami, G.,Lee, S.G. Korean Society of Industrial and Engineering Chemi 2017 Journal of industrial and engineering chemistry Vol.46 No.-
<P>s-DL4 is a variant of green fluorescent protein (GFP), exclusively deposited in vivo into active inclusion bodies (IBs). In this study, we demonstrated that s-DL4 is a self-aggregating molecule by performing structural analysis of s-DL4 IBs and studying in vivo/in vitro aggregating properties of the molecule. Fourier transform infrared analysis of IBs revealed that there were native GFP structures and intermolecular interactions between the protein molecules. s-DL4 was always deposited into insoluble intracellular IB aggregates, regardless of the protein expression rate. The active s-DL4 lBs dissolved in urea solution were aggregated and precipitated when the urea was removed by dialysis. (C) 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.</P>
Modulation of protein stability and aggregation properties by surface charge engineering
Raghunathan, Govindan,Sokalingam, Sriram,Soundrarajan, Nagasundarapandian,Madan, Bharat,Munussami, Ganapathiraman,Lee, Sun-Gu The Royal Society of Chemistry 2013 Molecular bioSystems Vol.9 No.9
<P>An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15–17) and s-GFP(+5–6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.</P> <P>Graphic Abstract</P><P>Engineering surface charge of proteins to modulate the properties of proteins by introducing surface charge mutations with and without stabilizing mutations. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c3mb70068b'> </P>
Characterization on the aggregation of self-aggregating green fluorescent protein variant
Govindan Raghunathan,Ganapathiraman Munussami,이선구 한국공업화학회 2017 Journal of Industrial and Engineering Chemistry Vol.46 No.-
s-DL4 is a variant of greenfluorescent protein (GFP), exclusively deposited in vivo into active inclusionbodies (IBs). In this study, we demonstrated that s-DL4 is a self-aggregating molecule by performingstructural analysis of s-DL4 IBs and studying in vivo/in vitro aggregating properties of the molecule. Fourier transform infrared analysis of IBs revealed that there were native GFP structures andintermolecular interactions between the protein molecules. s-DL4 was always deposited into insolubleintracellular IB aggregates, regardless of the protein expression rate. The active s-DL4 IBs dissolved inurea solution were aggregated and precipitated when the urea was removed by dialysis.
Govindan Raghunathan,Sriram Sokalingam,Nagasundarapandian Soundrarajan,Ganapathiraman Munussami,Bharat Madan,이선구 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.2
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.
A GFP variant exclusively deposited to fluorescent protein nanoparticles
( Govindan Raghunathan ),( Sriram Sokalingam ),( Ganapathiraman Munussami ),이선구 한국공업화학회 2014 한국공업화학회 연구논문 초록집 Vol.2014 No.1
Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science an nanotechnology. Here, we introduce a DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The intrinsic property of DL4 to form aggregates in vivo was exploited to produce fluorescent protein particles with different sizes. It is expected that DL4 IBs can be used as fluorescent biomaterials.