Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY

This paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the water flip back technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.     

Determination of h2JNN and h1JHN coupling constants across Watson–Crick base pairs in the Antennapedia homeodomain–DNA complex using TROSY

This paper describes NMR measurements of ¹⁵N–¹⁵N and ¹H–¹⁵N scalar couplings across hydrogen bonds in Watson–Crick base pairs, ^h2 J _NN and ^h1 J _HN, in a 17 kDa Antennapedia homeodomain–DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of ^h1 J _HN couplings in larger molecules. The ^h2 J _NN and ^h1 J _HN couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA beta-barrel from those of OmpX. Overall, the present data demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.     

Associations of Forest Type,Parasitism and Body Condition of Two European Passerines,Fringilla coelebs and Sylvia atricapilla

Human-induced forest modification can alter parasite-host interactions and might change the persistence of host populations. We captured individuals of two widespread European passerines (Fringilla coelebs and Sylvia atricapilla) in southwestern Germany to disentangle the associations of forest types and parasitism by haemosporidian parasites on the body condition of birds. We compared parasite prevalence and parasite intensity, fluctuating asymmetries, leukocyte numbers, and the heterophil to lymphocyte ratio (H/L-ratio) among individuals from beech, mixed-deciduous and spruce forest stands. Based on the biology of bird species, we expected to find fewer infected individuals in beech or mixed-deciduous than in spruce forest stands. We found the highest parasite prevalence and intensity in beech forests for F. coelebs. Although, we found the highest prevalence in spruce forests for S. atricapilla, the highest intensity was detected in beech forests, partially supporting our hypothesis. Other body condition or health status metrics, such as the heterophil to lymphocyte ratio (H/L-ratio), revealed only slight differences between bird populations inhabiting the three different forest types, with the highest values in spruce for F. coelebs and in mixed-deciduous forests for S. atricapilla. A comparison of parasitized versus non-parasitized individuals suggests that parasite infection increased the immune response of a bird, which was detectable as high H/L-ratio. Higher infections with blood parasites for S. atricapilla in spruce forest indicate that this forest type might be a less suitable habitat than beech and mixed-deciduous forests, whereas beech forests seem to be a suboptimal habitat regarding parasitism for F. coelebs.     

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site.     

Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

The interaction between the c(11)ring and the gammaepsilon complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c(11)ring and the gammaepsilon complex, with an apparent K(d) value of approximately 7.4nm. The rotor assembly was primarily dependent on the interaction of the cring with the gammasubunit, and binding of the cring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gammaE204, gammaF203 and epsilonH38) severely affected rotor assembly. The interaction kinetics between the gammaepsilon complex and c(11)ring mutants suggested that the assembly of the c(11)gammaepsiloncomplex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the cring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gammaE204 and epsilonH38, stabilized the holo-c(11)gammaepsilon complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the gamma subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F(1)complex to the rotor ring of F(o) to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F(1)F(o)complexes.     

Stochastic analysis of the GAL genetic switch in Saccharomyces cerevisiae: Modeling and experiments reveal hierarchy in glucose repression

Background

Most mathematical models of biochemical pathways consider either signalling events that take place within a single cell in isolation, or an 'average' cell which is considered to be representative of a cell population. Likewise, experimental measurements are often averaged over populations consisting of hundreds of thousands of cells. This approach ignores the fact that even within a genetically-homogeneous population, local conditions may influence cell signalling and result in phenotypic heterogeneity. We have developed a multi-scale computational model that accounts for emergent heterogeneity arising from the influences of intercellular signalling on individual cells within a population. Our approach was to develop an ODE model of juxtacrine EGFR-ligand activation of the MAPK intracellular pathway and to couple this to an agent-based representation of individual cells in an expanding epithelial cell culture population. This multi-scale, multi-paradigm approach has enabled us to simulate Extracellular signal-regulated kinase (Erk) activation in a population of cells and to examine the consequences of interpretation at a single cell or population-based level using virtual assays.

Results

A model consisting of a single pair of interacting agents predicted very different Erk activation (phosphorylation) profiles, depending on the formation rate and stability of intercellular contacts, with the slow formation of stable contacts resulting in low but sustained activation of Erk, and transient contacts resulting in a transient Erk signal. Extension of this model to a population consisting of hundreds to thousands of interacting virtual cells revealed that the activated Erk profile measured across the entire cell population was very different and may appear to contradict individual cell findings, reflecting heterogeneity in population density across the culture. This prediction was supported by immunolabelling of an epithelial cell population grown in vitro, which confirmed heterogeneity of Erk activation.

Conclusion

These results illustrate that mean experimental data obtained from analysing entire cell populations is an oversimplification, and should not be extrapolated to deduce the signal:response paradigm of individual cells. This multi-scale, multi-paradigm approach to biological simulation provides an important conceptual tool in addressing how information may be integrated over multiple scales to predict the behaviour of a biological system.     

Improved TROSY-HNCA experiment with suppression of conformational exchange induced relaxation

A general method for improving of the sensitivity of the TROSY-type triple resonance experiments in the presence of conformational exchange-induced (CSX) relaxation is proposed based on the use of CPMG-INEPT (Müller et al., J. Am. Chem. Soc., 1995, 117, 11043–11048) during the N–C polarization transfer periods. Significantly improved sensitivity is demonstrated for the majority of cross-peaks in the new [¹⁵N,¹H]-TROSY-XY-HNCA experiment, measured with partially folded RNase AS-Protein, with negligible loss of sensitivity for resonances unaffected by CSX relaxation. In addition, a comparison of cross-peak amplitudes in [¹⁵N,¹N]-TROSY-XY-HNCA and conventional [¹⁵N,¹H]-TROSY-HNCA spectra provides a quick and sensitive estimation of the CSX relaxation contribution.     

Two-dimensional NMR study of the conformation of (34–65)bacterioopsin polypeptide in SDS micelles

Summary The conformation of the synthetic 32-residue polypeptide, an analog of the membrane spanning segment B (residues 34-65) ofHalobacterium halobium bacteriobpsin, incorporated into perdeuterated sodium dodecyl sulfate micelles in the presence of trifluoroethanol was investigated by¹H NMR spectroscopy. The spectrum resonances were assigned by means of phase-sensitive DQF-COSY, TOCSY and NOESY techniques. Interproton nuclear Overhauser effects and deuterium exchange rates of individual NH groups were derived from two-dimensional NMR spectra. Analysis of the obtained data showed that segment B has a right-handed a-helical stretch from Lys⁴¹ to Leu⁶² with a kink at Pros⁵⁰. The-helix in the C-terminal part is terminated at Gly⁶³, which adopts a conformation typical of amino acid residues in a left-handed helix. The N-terminal part (residues 34–40) has no ordered conformation. NMR data are provided for comparison of the segment B conformation in the isotropic system of an organic solvent, in SDS micelles and in the purple membrane bacterioopsin. Factors affecting the conformation of membrane spanning segment B in various milieus are discussed.Dedicated to the memory of Professor V.F. Bystrov