Infrared studies of interaction between metal ions and Ca(2+)-binding proteins. Marker bands for identifying the types of coordination of the side-chain COO- groups to metal ions in pike parvalbumin (pI = 4.10).
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Metal-ligand interactions in the Ca(2+)-binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier-transform infrared spectroscopy. The region of the COO- antisymmetric stretch provides useful information on the types of coordination of the COO- groups to the metal ions in the Mg(2+)-, Mn(2+)-, and Ca(2+)-bound forms. In the spectrum of the Ca(2+)-bound form, two bands are observed at 1,582 and 1,553 cm-1, whereas, in the spectra of the Mg(2+)- and Mn(2+)-bound forms, bands are observed only in the region around 1,582 cm-1 and no band is found in the region around 1,553 cm-1. The 1,553-cm-1 band of the Ca(2+)-bound form reflects the bidentate coordination of the COO- groups of both Glu-62 in the CD site and Glu-101 in the EF site to the Ca2+ ions, which has been made clear by X-ray analysis as a feature of the Ca(2+)-bound form. Absence of such a band in the spectrum of the Mn(2+)-bound form is consistent with the X-ray structure of this form where both of the two COO- groups are unidentate. These unidentate COO- groups of Glu-62 and Glu-101 in the Mn(2+)-bound form seem to give rise to a band at 1,577-1,574 cm-1. The spectrum of the Mg(2+)-bound form is also consistent with the 'pseudo-bridging' coordination of the COO- group of Glu-101 reported in the X-ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the 'pseudo-bridging' coordination of the COO- group of Glu-62. (+info)
Assignment of 13C resonances and analysis of relaxation properties and internal dynamics of pike parvalbumin by 13C-NMR at natural abundance.
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Pike parvalbumin is an 11.5-kDa globular protein which binds Ca2+ through EF-hand structural motifs. Nearly complete assignment of the protonated 13C resonances has been achieved by means of heteronuclear two-dimensional experiments. The study shows that 13Ca chemical shifts can be very sensitive to localised conformational aspects. To characterise internal dynamics of pike parvalbumin, longitudinal-relaxation and transverse-relaxation rates and 1H-13C NOEs were measured for alpha-carbons at natural abundance by means of two-dimensional NMR spectroscopy. Relaxation data were obtained at a spectrometer frequency of 600 MHz for 69 residues with an even spread along the parvalbumin polypeptide chain. A double approach that included Lipari-Szabo analysis and direct mapping of spectral densities was used to interpret relaxation data in terms of internal dynamics. The former analysis provides valuable information about the overall rotational correlation time and S2 order parameters, while the mapping approach characterises the relative contributions of different motional frequencies. The results suggest that Ca(2+)-loaded pike parvalbumin has a rigid structure, even in the functional regions, i.e., the Ca(2+)-binding loops. The patterns of density-function values are more sensitive to the secondary structure than those of S2. Moreover, depending on the sampling frequency, these patterns reveal different aspects of structure-specific motions. (+info)
Historical analysis of genetic variation reveals low effective population size in a northern pike (Esox lucius) population.
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Effective population size (Ne) of a natural fish population was estimated from temporal changes in allele frequencies at seven microsatellite loci. Use of a historical collection of fish scales made it possible to increase the precision of estimates by increasing the time interval between samples and to use an equation developed for discrete generations without correcting for demographic parameters. Estimates of Ne for the time intervals 1961-1977 and 1977-1993 were 35 and 72, respectively. For the entire interval, 1961-1993, the estimate of Ne was 48 when based on a weighted mean derived from the above two estimates or 125 when calculated from 1961 and 1993 samples only. Corresponding ratios of effective size to adult census size ranged from 0.03 to 0.14. An Ne of 48 over a 32-year period would imply that this population lost as much as 8% of its heterozygosity in that time. Results suggest the potential for using genetic methods based on microsatellite loci data to compare historical trends in Ne with population dynamic parameters. Such comparisons will help to evaluate the relationship between genetic diversity and long-term persistence of natural populations. (+info)