title: Backbone dynamics and global effects of an activating mutation in minimized Mtu RecA inteins.
abstract: Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein DeltaDeltaIhh-V67CM and a single V67L mutant, DeltaDeltaIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites.
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Zhenming
authors.initials: Z
authors.lastname: Du
authors.forename: Yangzhong
authors.initials: Y
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Ban
authors.forename: Maria M
authors.initials: MM
authors.lastname: Lopez
authors.forename: Marlene
authors.initials: M
authors.lastname: Belfort
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D047668:Inteins / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D011938:Rec A Recombinases