The structural analysis of the redox complex between the soluble cytochrome c 552 and the membrane-integral cytochrome ba 3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c 552 and the Cu A domain, the fully functional water-soluble fragment of subunit II of the ba 3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [ 15 N, 1 H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c 552 and the Cu A domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c 552 to the Cu A center of the ba 3 oxidase has been predicted.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the k-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wildtype Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28 fi Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two welldefined a-helices (residues 5-11 and 18-27) and five b-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1 H 2 O-2 H 2 O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage k excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical Ôwinged-helixÕ DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the k Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.Keywords: excisionase; NMR spectroscopy; protein stability; isoaspartyl linkage; cis-proline.Knowledge about the molecular mechanisms of viral sitespecific integration/excision in prokaryotes and eukaryotes can be widely employed in biotechnological and medical applications, such as site-specific genomic targeting, drug design, vector construction, etc. The structural determinants of integrative recombination were studied extensively in various viruses during the 1990s [1][2][3][4], and almost all proteins participating in the bacteriophage k recombination system have been structurally characterized [5][6][7][8]. However, very little is known about the structural basis of excisionase function, although its importance is generally recognized.Two closely related bacteriophages -k and HK022 -use common mechanisms for integration/excision of their genomes during a life cycle. The phage-encoded integrase, Int, recognizes attP (on the phage chromosome) and attB (on the bacterial chromosome) core sites and performs sitespecific recombination with the help of the cell-encoded integration host factor, IHF, resulting in integration of the circular phage DNA into the cellular chromosome. The attP and attB sites generate the prophage sites attR and attL, which flank the inserted phage DNA. The reverse reaction (...

An allotypic variation at position 25 influences the fibrillogenicity of lambdaVI light chains, which are related to humoral immune response and have been associated with AL amyloidosis. The full resonance assignment and a preliminary structural characterization of 6aJL2(R25G) are reported.

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