Abstract
Parvalbumin (PV) is an intracellular cation binding protein containing the helix-loop-helix divalent binding domain characteristic of the large EF hand super family of proteins. Parvalbumin was the first EF hand protein to be structurally characterized and is the model for understanding other EF hand proteins including troponin C, myosin light chain, and calmodulin. PV facilitates muscle relaxation by sequestering Ca2+ and has not yet been well studied in elasmobranch fish. The Atlantic stingray, Dasyatis sabina¸ found along the Gulf of Mexico and southeastern Atlantic coasts, is euryhaline and is able to compensate for changing environmental salinity by altering plasma and intracellular solutes, primarily urea and the counteracting methylamines (betaine and TMAO). Compensation to changes in salinity of the habitat impacts the solute environment of intracellular proteins like PV. Thus, determining the impact of changes in in situ concentrations of these organic osmolytes on PV function in marine and freshwater acclimatized populations of D. sabina could provide insight into intracellular correlates of euryhaline tolerance for this species. Parvalbumins from both marine and freshwater D. sabina and the major PV isoform (II) from the freshwater teleost Cyprinus carpio (carp) were purified by gel permeation and DEAE chromatography. Purity of preparations was confirmed by SDS PAGE and western blot analysis. Direct sequencing of amino acids of PVs from D. sabina further validated that these proteins were true PVs. Both populations of D. sabina exhibited two PV isoforms, a major (~12.18 kDa, PV I) and a minor (11.96 kDa, PV II). In addition to PVs isolated from muscle, the full length cDNA for D. sabina PV I was inserted into an expression vector. Recombinant PV I was expressed, purified and assessed for purity. This recombinant PV I was used in some phases of the experimental work. The two PV isoforms from D. sabina displayed significantly different responses to urea, betaine and TMAO. The most interesting result was the observation of a significant increase in Ca2+ binding affinity of PV II in the presence of urea, a protein destabilizer. In contrast, PV I’s ability to bind Ca2+ and Mg2+ was found to be insensitive to the effects of urea and TMAO. For the most part, cation binding in Carp II PV was found to be insensitive to concentrations of organic osmolytes prevailing in the freshwater ray. Thus, it appears that organic osmolytes have isoform specific effects on PV function. A study of the impact of increasing urea concentrations on cation binding revealed that PV I lost complete ion binding ability at 3.6 M urea. Unidirectional rate constants for cation binding were determined by stopped flow analysis. Interestingly, the major PV isoform from both D. sabina and carp showed faster on rates (kon) with minimal effects on koff for Ca2+ and Mg2+ in the presence of concentrations of urea and TMAO comparable to physiological conditions present in marine rays. In hopes of better understanding how physiologically relevant concentrations of urea and TMAO affect the stability of PV, structural studies were performed using circular dichroism (CD) spectrometry. CD studies showed a significant decrease in -helical structure for D. sabina PVI and carp II PV in the presence of increasing urea concentrations suggesting a decrease in protein stability. A shift toward a more random coiled structure was also seen in the absence of Ca2+. Environmental changes, including osmolarity, can profoundly influence muscle protein functionality and an understanding of the impact of these changes may provide insight into how PV and other EF hand proteins are able to retain function under stressful environmental conditions. This study has found that the functional properties of PVs appear to be fairly resilient to the effects of urea, betaine and TMAO, at least when exposed to physiological concentrations. However, while these organic osmolytes may not be significantly altering PV’s ability to function properly, there is a dramatic effect on the overall structural stability. In conclusion, this effort has shown that the euryhaline stingray Dasyatis sabina has two PV isoforms which differ somewhat in the impact of organic solutes on cation binding. The most thoroughly studied isoform, PV I, displays functional and structural responses to organic osmolytes consistent with the euryhaline behavior of this stingray.
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