title: Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.
abstract: In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico.|Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico.
authors.forename: Devin W
authors.initials: DW
authors.lastname: Close
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia.
authors.forename: Craig Don
authors.initials: CD
authors.lastname: Paul
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico.
authors.forename: Patricia S
authors.initials: PS
authors.lastname: Langan
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia.
authors.forename: Matthew C J
authors.initials: MC
authors.lastname: Wilce
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia.
authors.forename: Daouda A K
authors.initials: DA
authors.lastname: Traore
authors.affiliation: Department of Biochemistry, UT Southwestern Medical Center, Dallas, Texas.
authors.forename: Randal
authors.initials: R
authors.lastname: Halfmann
authors.affiliation: Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico.
authors.forename: Reginaldo C
authors.initials: RC
authors.lastname: Rocha
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico.
authors.forename: Geoffery S
authors.initials: GS
authors.lastname: Waldo
authors.affiliation: Integral Molecular, Philadelphia, Pennsylvania.
authors.forename: Riley J
authors.initials: RJ
authors.lastname: Payne
authors.affiliation: Integral Molecular, Philadelphia, Pennsylvania.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Rucker
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia.
authors.forename: Mark
authors.initials: M
authors.lastname: Prescott
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico.
authors.forename: Andrew R M
authors.initials: AR
authors.lastname: Bradbury
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry; D001681:Biological Assay; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D055672:Static Electricity
chemical_list: D000682:Amyloid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: GFP structure; X-ray crystallography; eCGP123; protein engineering; structure-guided mutagenesis; thermal green protein; thermal stability