title: Chimeric Avidin--NMR structure and dynamics of a 56 kDa homotetrameric thermostable protein.
abstract: Chimeric avidin (ChiAVD) is a product of rational protein engineering remarkably resistant to heat and harsh conditions. In quest of the fundamentals behind factors affecting stability we have elucidated the solution NMR spectroscopic structure of the biotin-bound form of ChiAVD and characterized the protein dynamics through 15N relaxation and hydrogen/deuterium (H/D) exchange of this and the biotin-free form. To surmount the challenges arising from the very large size of the protein for NMR spectroscopy, we took advantage of its high thermostability. Conventional triple resonance experiments for fully protonated proteins combined with methyl-detection optimized experiments acquired at 58°C were adequate for the structure determination of this 56 kDa protein. The model-free parameters derived from the 15N relaxation data reveal a remarkably rigid protein at 58°C in both the biotin-bound and the free forms. The H/D exchange experiments indicate a notable increase in hydrogen protection upon biotin binding.
authors: Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors: Helena
authors: H
authors: Tossavainen
authors: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland; Fimlab Laboratories, Tampere, Finland.
authors: Sampo
authors: S
authors: Kukkurainen
authors: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland; BioMediTech, Tampere, Finland; Fimlab Laboratories, Tampere, Finland.
authors: Juha A E
authors: JA
authors: Määttä
authors: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland; BioMediTech, Tampere, Finland.
authors: Niklas
authors: N
authors: Kähkönen
authors: Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors: Tero
authors: T
authors: Pihlajamaa
authors: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland; BioMediTech, Tampere, Finland; Fimlab Laboratories, Tampere, Finland.
authors: Vesa P
authors: VP
authors: Hytönen
authors: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland; BioMediTech, Tampere, Finland.
authors: Markku S
authors: MS
authors: Kulomaa
authors: Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
authors: Perttu
authors: P
authors: Permi
mesh_terms: D001360:Avidin / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D013816:Thermodynamics
chemical_list: D001360:Avidin; D001710:Biotin