PubMed · 6902318
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S Hopkins. 1980. Calcium.. https://pubmed.ncbi.nlm.nih.gov/6902318/
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Velocity sedimentation experiments using authentic fibrillin-1 demonstrated sedimentation coefficients of s20,w0 = 5.1 +/- 0.1 in the Ca2+ form and s20,w0 = 6.2 +/- 0.1 in the Ca2+-free form. Calculations based on these results and the corresponding molecular mass predicted a shortening of fibrillin by approximately 25% and an increase in width of approximately 13-17% upon removal of Ca2+. These observations were confirmed by analysis of Ca2+-loaded and Ca2+-free rotary shadowed fibrillin molecules. Analysis of recombinant fibrillin-1 subdomain rF17, consisting primarily of an array of 12 Ca2+-binding epidermal growth factor (cbEGF)-like repeats, by analytical ultracentrifugation and rotary shadowing further confirmed Ca2+-dependent structural changes in the tertiary structure of fibrillin-1. Based on these results, the contribution of a single cbEGF-like repeat to the length of tandem arrays is predicted to be approximately 3 nm in the Ca2+ form. Ca2+-free forms demonstrated a decrease of 20-30% in length, indicating significant structural changes of these motifs when they occur in tandem. Circular dichroism measurements of rF17 in the presence and absence of Ca2+ indicated secondary structural changes within and adjacent to the interdomain regions that connect cbEGF-like repeats. The results presented here suggest a flexible structure for the Ca2+-free form of fibrillin which becomes stabilized, more extended, and rigid in the Ca2+ form.
Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.
Previous reports have suggested that receptors for immunoglobulin G (IgG), FcgammaRs, directly activate a nonselective cation channel (Young, J. D.-E., Unkeless, J. C., Young, T. M., Mauro, A., and Cohn, Z. A. (1983) Nature 306, 186-189; Nelson, D. J., Jacobs, E. R., Tang, J. M., Zeller, J. M., and Bone, R. C. (1985) J. Clin. Invest. 76, 500-507). To investigate the mechanisms underlying membrane conductance changes following human high affinity (FcgammaRI) receptor activation, we have used the human monocytic cell line U937 and combined conventional whole cell patch-clamp recordings with single cell fura-2 Ca2+ measurements. Using a K+-free internal solution, antibody cross-linking of IgG-occupied FcgammaRI activated an inward current at negative potentials, whose amplitude and time course mirrored the concomitant rise in intracellular Ca2+. Current-voltage relationships, obtained under different ionic conditions, revealed a monovalent cation-selective conductance that, under physiological conditions, would result in Na+ influx. Noise analysis of current recordings indicated a single channel conductance of 18 picosiemens and a mean opening time of 4.5 ms. This current was also activated by rises in intracellular Ca2+ induced by ionomycin (3 microM) or thapsigargin (1 microM). Addition of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid to the intracellular medium abolished any channel activation by ionomycin, FcgammaRI, or the low affinity receptor, FcgammaRII. These results demonstrate that FcgammaRI activation triggers a novel Ca2+-activated channel selective for monovalent cations and that neither FcgammaRI nor FcgammaRII can directly activate a channel.