1H, 13C, and 15N resonance assignment of Bombyx mori chemosensory protein 1 (BmorCSP1).
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Animals↗
Biomedical subjects
Lukás Zídek
Publications and source records attributed to Lukás Zídek.
Molecular dynamics study of major urinary protein-pheromone interactions: a structural model for ligand-induced flexibility increase.
Recently, two independent (15)N NMR relaxation studies indicated that in contrast to the decreased flexibility expected for induced-fit interactions, the backbone flexibility of major urinary protein isoform I (MUP-I) slightly increased upon complex formation with its natural pheromone 2-sec-butyl-4,5-dihydrothiazol. We have investigated the subtle details of molecular interactions by molecular dynamics simulations in explicit solvent. The calculated order parameters S(2) for a free- and ligand-bound protein supply evidence that mobility in various regions of MUP-I can be directly related to small conformational changes of the free- and complexed protein resulting from modifications of the hydrogen bonding network.
Computer Simulation↗
Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2- sec-butyl-4,5-dihydrothiazole.
Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by (15)N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2- sec -4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature dependence of the motional parameters.
Animals↗
Internal consistency of NMR data obtained in partially aligned biomacromolecules.
A method of testing structure-related NMR data prior to structure calculations is presented. The test is applicable to second rank tensor interactions (dipolar coupling, anisotropic chemical shielding, and quadrupolar interaction) observed in partially aligned samples of biomacromolecules. The method utilizes the fact that only limited number of frequencies corresponding to the mentioned interactions can be measured independently in a rigid fragment of the macromolecule. Additional values can be predicted as linear combinations of the set of independent frequencies. Internal consistency of sufficiently large sets of frequencies measured in individual molecular fragments is tested by comparing the experimental data with their predicted values. The method requires only knowledge of local geometry (i.e., definition of the interaction tensors in the local coordinate frames of the fragments). No information about the alignment or shape of the molecule is required. The test is best suited for planar fragments. Application to peptide bonds and nucleic acid bases is demonstrated.
Anisotropy↗
Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings.
The structure of the 13C,15N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond 1H-13C, 1H-15N, 13C-13C and 13C-15N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.
Base Sequence↗
Pheromone binding by polymorphic mouse major urinary proteins.
Mouse major urinary proteins (MUPs) have been proposed to play a role in regulating the release and capture of pheromones. Here, we report affinity measurements of five recombinant urinary MUP isoforms (MUPs-I, II, VII, VIII, and IX) and one recombinant nasal isoform (MUP-IV) for each of three pheromonal ligands, (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (+/-)dehydro-exo-brevicomin (DHB). Dissociation constants for all MUP-pheromone pairs were determined by isothermal titration calorimetry, and data for SBT were corroborated by measurements of intrinsic protein fluorescence. We also report the isolation of MUP-IV protein from mouse nasal extracts, in which MUP-IV mRNA has been observed previously. The affinity of each MUP isoform for SBT (K(d) approximately 0.04 to 0.9 micro M) is higher than that for DHB (K(d) approximately 26 to 58 micro M), which in turn is higher than that for HMH (K(d) approximately 50 to 200 micro M). Isoforms I, II, VIII, and IX show very similar affinities for each of the ligands. MUP-VII has approximately twofold higher affinity for SBT but approximately twofold lower affinity for the other pheromones, whereas MUP-IV has approximately 23-fold higher affinity for SBT and approximately fourfold lower affinity for the other pheromones. The variations in ligand affinities of the MUP isoforms are consistent with structural differences in the binding cavities of the isoforms. The data indicate that the concentrations of available pheromones in urine may be influenced by changes in the expression levels of urinary MUPs or the excretion levels of other MUP ligands. The variation in pheromone affinities of the urinary MUP isoforms provides only limited support for the proposal that MUP heterogeneity plays a role in regulating profiles of available pheromones. However, the binding data support the proposed role of nasal MUPs in sequestering pheromones and possibly transporting them to their receptors.
Amino Acid Sequence↗