A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables...

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Bibliographic Details
Published in:PloS one 2012-07, Vol.7 (7), p.e40410
Main Authors: Sokalingam, Sriram, Raghunathan, Govindan, Soundrarajan, Nagasundarapandian, Lee, Sun-Gu
Format: Article
Language:English
Subjects:
Algorithms
Amino Acid Motifs
Amino Acid Substitution
Amino acids
Arginine
Arginine - chemistry
Arginine - genetics
Biochemistry
Biology
Biosensors
Bridges
Bridges (Structures)
Chemical engineering
Chemistry
Comparative analysis
Detergents
Detergents - chemistry
Electrostatic properties
Fluorescence
Green fluorescent protein
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - genetics
Half-Life
Humicola insolens
Hydration
Hydrogen
Hydrogen Bonding
Hydrogen bonds
Hydrogen-Ion Concentration
Ionic interactions
Lysine
Lysine - chemistry
Lysine - genetics
Models, Molecular
Molecular biology
Mutagenesis
Mutagenesis, Site-Directed
Physiology
Protein Denaturation
Protein engineering
Protein folding
Protein kinase A
Protein Stability
Protein structure
Protein Structure, Tertiary
Proteins
Quaternary Ammonium Compounds - chemistry
Rigidity
Salt
Salts
Sodium Dodecyl Sulfate - chemistry
Spectrometry, Fluorescence
Structural stability
Surface Properties
Surfactants
Thermal stability
Urea
Urea - chemistry
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Summary:Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0040410