title: Isoforms of green fluorescent protein differ from each other in solvent molecules 'trapped' inside this protein.
abstract: Green fluorescent protein (GFP) has been studied quite thoroughly, however, up to now some experimental data have not been explained explicitly. For example, under native conditions this protein can have two isoforms differing in their mobility in gel. In this case, no differences between the isoforms are revealed under denaturing conditions. In order to understand the difference in the isoforms of this protein, we have investigated GFP-cycle3 using mass spectrometry, gel electrophoresis, size exclusion chromatography, microcalorimetry, and spectroscopy methods under varying conditions. We have also designed and studied three mutant forms of this protein with substitutions of amino acid residues inside the GFP barrel. The mutations have allowed us to influence the formation of different GFP isoforms. Each of the mutant proteins has predominantly only one isoform. As a result of the performed research, it can be concluded that most likely the GFP isoforms differ in the solvent molecules 'trapped' inside the GFP barrel. In their turn, these molecules have an effect on the protein charge and consequently on its mobility at electrophoresis under native conditions.
authors.affiliation: a Institute of Protein Research , Russian Academy of Sciences , 142290 Pushchino , Moscow Region , Russia.
authors.forename: Kseniya F
authors.initials: KF
authors.lastname: Glukhova
authors.affiliation: a Institute of Protein Research , Russian Academy of Sciences , 142290 Pushchino , Moscow Region , Russia.
authors.forename: Victor V
authors.initials: VV
authors.lastname: Marchenkov
authors.affiliation: a Institute of Protein Research , Russian Academy of Sciences , 142290 Pushchino , Moscow Region , Russia.
authors.forename: Tatiana N
authors.initials: TN
authors.lastname: Melnik
authors.affiliation: a Institute of Protein Research , Russian Academy of Sciences , 142290 Pushchino , Moscow Region , Russia.
authors.forename: Bogdan S
authors.initials: BS
authors.lastname: Melnik
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D063347:Native Polyacrylamide Gel Electrophoresis; D011489:Protein Denaturation; D020033:Protein Isoforms; D012997:Solvents / Q000737:chemistry*; D044366:Transition Temperature
chemical_list: D020033:Protein Isoforms; D012997:Solvents; D049452:Green Fluorescent Proteins
keywords: green fluorescent protein; irreversible denaturation; isoforms; native polyacrylamide gel electrophoresis