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Ns-?s Time-Resolved Step-Scan FTIR of ba? Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927.

ABSTRACT: Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of the ba? oxidoreductase from Thermus thermophilus in the ns-?s time range and in the pH/pD 6-9 range. The data revealed a pH/pD sensitivity of the D372 residue and of the ring-A propionate of heme a?. Based on the observed transient changes a model in which the protonic connectivity of w941-w946-927 to the D372 and the ring-A propionate of heme a? is described.

SUBMITTER: Nicolaides A

PROVIDER: S-EPMC5085690 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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ns-μs Time-Resolved Step-Scan FTIR of ba₃ Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927.

Nicolaides Antonis A Soulimane Tewfik T Varotsis Constantinos C

International journal of molecular sciences 20160929 10

Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of the ba₃ oxidoreductase from Thermus thermophilus in the ns-μs time range and in the pH/pD 6-9 range. The data revealed a pH/pD sensitivity of the D372 residue and of the ring-A propionate of heme a₃. Based on the observed transient changes a model in which the protonic connectivity of w941-w946-927 to the D372 and the ring-A propionate of heme a₃ is described. ...[more]

PMID: 27690021

Similar Datasets

Spectroscopic and kinetic investigation of the fully reduced and mixed valence states of ba3-cytochrome c oxidase from Thermus thermophilus: a Fourier transform infrared (FTIR) and time-resolved step-scan FTIR study.

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Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus.

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Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy.

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Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(3) cytochrome oxidases.

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An unexpected outcome of surface engineering an integral membrane protein: improved crystallization of cytochrome ba(3) from Thermus thermophilus.

Project description:Past work has shown that it is feasible to mutate surface residues of soluble proteins and to a lesser extent membrane proteins in order to improve their crystallization behavior. Described here is a successful application of this approach to the integral membrane protein Thermus thermophilus cytochrome ba(3) oxidase. Two mutant forms of this enzyme (I-K258R and I-K258R/II-E4Q) were created in which symmetrical crystal contacts within crystals of wild-type enzyme were modified. These mutant proteins had greatly shortened crystallization times, decreasing from approximately 30 d for the wild type to 1-3 d for the mutants, and crystallization was highly reproducible. Native-like proteins crystallize in space group P4(3)2(1)2, whereas the mutant proteins crystallize in space group P4(1)2(1)2 with a different packing arrangement. Crystals of the P4(3)2(1)2 form occasionally diffracted to 2.4-2.3 A resolution following controlled dehydration, while those of the P4(1)2(1)2 form routinely diffracted to between 3.0 and 2.6 A for crystals that had been cryoprotected but not dehydrated.

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Thiostrepton-resistant mutants of Thermus thermophilus.

Project description:Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle components that mediate interactions of translation factors with the ribosome. This site is also the target of the antibiotic thiostrepton, which has been proposed to act by preventing important structural transitions that occur in this region of the ribosome during protein synthesis. Here, we describe the isolation and characterization of spontaneous thiostrepton-resistant mutants of the extreme thermophile, Thermus thermophilus. All mutations were found at conserved positions in the flexible N-terminal domain of L11 or at conserved positions in the L11-binding site of 23S rRNA. A number of the mutant ribosomes were affected in in vitro EF-G-dependent GTP hydrolysis but all showed resistance to thiostrepton at levels ranging from high to moderate. Structure probing revealed that some of the mutations in L11 result in enhanced reactivity of adjacent rRNA bases to chemical probes, suggesting a more open conformation of this region. These data suggest that increased flexibility of the factor binding site results in resistance to thiostrepton by counteracting the conformation-stabilizing effect of the antibiotic.

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