Birch pollinosis is one of the prevailing allergic diseases. In all, 5 -20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca 2þ -binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal a-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca 2þ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.

The carboxy-terminal domain of the transcription factor Escherichia coli NusA, NusACTD, interacts with the protein N of bacteriophage l, lN, and the carboxyl terminus of the E. coli RNA polymerase a subunit, aCTD. We solved the solution structure of the unbound NusACTD with high-resolution nuclear magnetic resonance (NMR). Additionally, we investigated the binding sites of lN and aCTD on NusACTD using NMR titrations. The solution structure of NusACTD shows two structurally similar subdomains, NusA and , matching approximately two homologous acidic sequence repeats. Further characterization of NusACTD with 15 N NMR relaxation data suggests that the interdomain region is only weakly structured and that the subdomains are not interacting. Both subdomains adopt an (HhH) 2 fold. These folds are normally involved in DNAprotein and protein-protein interactions. NMR titration experiments show clear differences of the interactions of these two domains with aCTD and lN, in spite of their structural similarity.Keywords: NusA; anti-termination; termination; NMR; RNA polymerase; N-protein; phage l; HhH motif RNA synthesis in Escherichia coli is catalyzed by RNA polymerase (RNAP), a multiprotein enzyme whose core shows an a 2 bb 0 subunit composition (Nudler 1999). After initiation of transcription, the essential transcription factor NusA (N utilization substance A) associates with the RNAP core enzyme, where it modulates transcriptional pausing, termination, and anti-termination (Liu et al. 1996).The crystal structures of two non-E. coli NusA factors have been solved so far (Thermotoga maritima [Worbs et al. 2001;Shin et al. 2003], Mycobacterium tuberculosis [Gopal et al. 2001]). These structures show a common domain organization, that is, an amino-terminal RNAPbinding domain, followed by one S1 and two KH (K homology) RNA-binding domains. This NusA core organization is conserved in all bacteria for which such sequence information is available. An additional carboxyterminal region, NusACTD, comprising $160 residues is found in several a-, b-, and g-proteobacteria like the enterobacterium E. coli, as well as in Chlamydia or Treponema (Mah et al. 2000). Though NusACTD is not as highly conserved as the NusA core, the region is characterized by its acidity and frequently by an internal sequence repeat of $70 residues. Reprint requests to: Paul Ro¨sch, Department of Biopolymers, Universita¨tsstr. 30, University of Bayreuth, 95440 Bayreuth, Germany;.Abbreviations: aCTD, carboxy-terminal domain of the a subunit of the RNAP; E. coli, Escherichia coli; HetNOE, f 1 Hg 15 N heteronuclear NOE; HhH, helix hairpin helix; HSQC, heteronuclear single quantum coherence; KH, K homology; lN, protein N of phage l; NOESY, nuclear Overhauser spectroscopy; NusA, N utilization substance A; NusACTD, carboxy-terminal domain of NusA; NMR, nuclear magnetic resonance, PG-SLED, pulse gradient-stimulated echo longitudinal encode-decode; RDC, residual dipolar coupling; RMSD, root meansquare deviation; RNAP, RNA polymerase; SAM, sterile a motif.Arti...

N protein of the Escherichia coli phage lambda (lambdaN) is involved in antitermination, a transcription regulatory process that is essential for the expression of delayed early genes during phage lytic development. lambdaN is an intrinsically unstructured protein that possesses three distinct binding sites interacting with the carboxy terminus of the E. coli host factor protein NusA, the viral nutBoxB-RNA, and RNA polymerase, respectively. Heteronuclear NMR experiments with lambdaN(1-53) in complex with NusA(339-495) revealed that upon complex formation the lambdaN-binding interface, lambdaN(34-47), adopts a rigid structure. NMR data also indicate the induction of a weak helical structure in the nutboxB RNA-binding region lambdaN(1-22) upon binding to NusA(339-495) even in the absence of RNA. Titration experiments of the lambdaN(1-53)-nutBoxB RNA complex with NusA(339-495) revealed that the ternary complex can be described in terms of two structurally independent binary interactions. Furthermore, chemical-shift perturbation experiments with different NusA constructs and different lambdaN peptides showed that only NusA(353-416) is involved in lambdaN binding. We found that only one molecule of NusA(339-426) binds to one molecule of lambdaN(1-53). We also clarified the role of the lambdaN-binding region and could show that N41-R47 also binds to NusA(339-495). Furthermore, we observe that lambdaN(1-22) adopts a helical fold upon binding to NusA(339-495), in agreement with one of the theoretical models of lambdaN action.

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Summary. Accurate estimations of experimental uncertainties of relaxation rates are of vital importance for the interpretation of relaxation data, in particular for the Lipari-Szabo 'model free' approach and for comparative relaxation studies. Here we report a systematic investigation of different methods for the estimation of experimental uncertainties on longitudinal R 1 and transversal R 2 rates using two different schemes for sampling the rates. We show that certain combinations of sampling strategies and methods of estimating experimental uncertainties result in wrong rates and rate errors. Practical recommendations for obtaining proper rate and rate uncertainties are deduced.