title: Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.
abstract: Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Anna V
authors.initials: AV
authors.lastname: Lomonosova
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Ovchinnikova
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Alexei S
authors.initials: AS
authors.lastname: Kazakov
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia; Department of Biosciences, Åbo Akademi University, Turku, 20520, Finland.
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Denesyuk
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Sofin
authors.affiliation: Antherix, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Roman V
authors.initials: RV
authors.lastname: Mikhailov
authors.affiliation: Antherix, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Andrei B
authors.initials: AB
authors.lastname: Ulitin
authors.affiliation: Antherix, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia; Biomirex Inc., 304 Pleasant Street, Watertown, Massachusetts, 02472, United States of America.
authors.forename: Tajib A
authors.initials: TA
authors.lastname: Mirzabekov
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Permyakov
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya str., 7, Pushchino, Moscow region, 142290, Russia.
authors.forename: Sergei E
authors.initials: SE
authors.lastname: Permyakov
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019791:Guanidine / Q000737:chemistry; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D015202:Protein Engineering; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D043902:Pyrococcus horikoshii / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054748:Ribosome Subunits, Large, Archaeal / Q000737:chemistry / Q000378:metabolism
chemical_list: D000906:Antibodies; D019843:Archaeal Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D019791:Guanidine