Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins
VILIP and NCS-1, neural-specific, 22-kDa Ca2+-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties.Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2' binding) revealed that both recombinant proteins possess only t...
محفوظ في:
| المؤلفون الرئيسيون: | , , , , , , |
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| التنسيق: | Artigo |
| اللغة: | الإنجليزية |
| منشور في: |
1994
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| الوصول للمادة أونلاين: | https://doi.org/10.1016/s0021-9258(20)30063-6 |
| الوسوم: |
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| الملخص: | VILIP and NCS-1, neural-specific, 22-kDa Ca2+-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties.Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2' binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca" or M e .VILIP binds cations without cooperativity with intrinsic affinity constants K c , of 1.0 x los M -~ and KfMg of 4.8 x lo3 M-'.Mg2' antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape.The competition equation yields a Kt%, camp value of 180 for both sites.NCS-1 binds two M e without cooperativity with K M g of 8.3 x lo4 and two Ca2+ with very strong positive cooperativity (n, = 1.96).In the absence of Mg2' the Real and K C a values are 8.9 x lo4 and 1.4 x lo8 "I, respectively, which represent an allosteric increase of 1600-fold.M e shifts the Ca2+-binding isotherms to higher Ca2+ concentrations, yielding a KMg, camp value of 800 M" for both sites.Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2' binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2'/Ca2+ antagonism is much weaker than in NCS-1.Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured.Ca2+ binding promotes a 20430% increase of fluorescence in both proteins, but whereas the Mg2' form of VILIP has the same fluorescence properties as the metal-free form, Me-saturated NCS-1 has those of the CaZ+ form.Near W difference spectra confirmed that in VILIP the M e form is very similar to the metalfree form; in NCS-1 it is different, especially in the Tyr region.NCS-1 possesses one unique Cys-38 in EF-hand site I.Its reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca2+-and M eloaded protein, but It,, is 4-fold higher in metal-free NCS-1.VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein.The reactivity of the two accessible thiols is identical in the metal-free and M e forms and 5-fold higher than in the Ca2+ form.In NCS-1 the accessibility of the hydrophobic |
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