Luminescence and circular-dichroism analysis of terbium binding by pig intestinal calcium-binding protein (relative mass = 9000)

1984 ◽  
Vol 62 (6) ◽  
pp. 434-442 ◽  
Author(s):  
Joe D. J. O'Neil ◽  
Keith J. Dorrington ◽  
Theo Hofmann
Keyword(s):  
Circular Dichroism ◽  
Calcium Binding ◽  
Binding Protein ◽  
Three Dimensional ◽  
Cation Binding ◽  
Dimensional Structure ◽  
Relative Mass ◽  
Excitation Spectra ◽  
Fluorescence Excitation ◽  
Circular Dichroïsm

The structure and conformations of pig intestinal Ca-binding protein (CaBP) have been studied by terbium luminescence enhancement and circular dichroism. The two cation-binding sites bind Tb3+ sequentially; the affinity of the first site is > 107 M−1 and the affinity of the second site is approximately 105 M−1. Filling of the first site enhances the fluorescence of the single tyrosine residue, whereas Tb3+ in the second site quenches the fluorescence. Excitation spectra of the Tb3+-bound forms of CaBP show that considerable energy transfer takes place from phenylalanine residues to the bound Tb3+, although some transfer from tyrosine is also detected. The sequence in which the sites are filled was deduced from these results and the published three-dimensional structure of the cow intestinal CaBP. Tb3+ bound approximately 20 Å (1 Å = 0.1 nm) from the tyrosine induced a large increase in the optical activity of this residue. We argue that a potentially important conformational change is induced in CaBP by cation binding.

Spectroscopic studies on the binding of divalent cations to porcine intestinal calcium-binding protein

1978 ◽  
Vol 56 (6) ◽  
pp. 492-499 ◽  
Author(s):  
K. J. Dorrington ◽  
D. I. C. Kells ◽  
A. J. W. Hitchman ◽  
J. E. Harrison ◽  
T. Hofmann
Keyword(s):  
Optical Activity ◽  
Calcium Binding ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Cation Binding ◽  
Side Chain ◽  
Difference Spectroscopy ◽  
High Affinity ◽  
Circular Dichroïsm ◽  
Circular Dichroic

Circular dichroism and ultraviolet absorption difference spectroscopy have been used to study the binding of a series of divalent and trivalent cations to porcine intestinal calcium-binding protein (CaBP). When calcium is bound to the single high-affinity site on CaBP, the aromatic optical activity is greatly increased. Analysis of the circular dichroic spectra, obtained in the presence and absence of calcium, suggested that although changes in the optical activity of the single tyrosyl residue accounted for much of the overall change observed upon binding calcium, one or more of the five phenylalanyl residues was also perturbed. All the cations tested, with the exception of lead which gave rise to unique spectral effects, caused the same changes in optical activity between 300 and 250 nm. In the peptide absorption region, CaBP exhibited optical activity typical of an α-helical protein and no significant changes were observed in the presence of any of the cations tested. Cation-binding curves obtained from the circular dichroic data for the cations bound with high affinity (i.e., calcium, strontium, and the trivalent lanthanide ions) showed that the apparent number of binding sites was inversely related to the protein concentration. This phenomenon was accounted for by the concentration-dependent aggregation of CaBP observed in earlier studies. The binding data, obtained using circular dichroism, clearly indicated that the affinity of CaBP for the various cations was related to their ionic radius. Absorption difference spectra were observed when calcium was bound to CaBP. The features of these spectra confirmed that phenylalanyl as well as tyrosyl transitions were perturbed upon calcium binding. The extent to which the tyrosyl side chain was exposed to solvent was determined by solvent perturbation difference spectroscopy using perturbing agents of differing molecular radius. The apparent degree of exposure increased as the perturbant size decreased suggesting that the side chain was located in a cleft. Bound calcium did not change the degree of exposure. These data, together with complementary data obtained with bovine CaBP, were discussed in terms of the geometry of the cation-binding site.

scholarly journals Mass-resolved electronic circular dichroism ion spectroscopy

Science ◽  
2020 ◽  
Vol 368 (6498) ◽  
pp. 1465-1468 ◽  
Author(s):  
Steven Daly ◽  
Frédéric Rosu ◽  
Valérie Gabelica
Keyword(s):  
Circular Dichroism ◽  
Three Dimensional ◽  
Polarized Light ◽  
Negative Ions ◽  
Data Interpretation ◽  
Electronic Circular Dichroism ◽  
Circularly Polarized Light ◽  
Chiral Compounds ◽  
Electron Photodetachment ◽  
Circular Dichroïsm

DNA and proteins are chiral: Their three-dimensional structures cannot be superimposed with their mirror images. Circular dichroism spectroscopy is widely used to characterize chiral compounds, but data interpretation is difficult in the case of mixtures. We recorded the electronic circular dichroism spectra of DNA helices separated in a mass spectrometer. We studied guanine-rich strands having various secondary structures, electrosprayed them as negative ions, irradiated them with an ultraviolet nanosecond optical parametric oscillator laser, and measured the difference in electron photodetachment efficiency between left and right circularly polarized light. The reconstructed circular dichroism ion spectra resembled those of their solution-phase counterparts, thereby allowing us to assign the DNA helical topology. The ability to measure circular dichroism directly on biomolecular ions expands the capabilities of mass spectrometry for structural analysis.

Novel clostridial cell-surface hemicellulose-binding CBM3 proteins

2021 ◽  
Vol 77 (4) ◽  
Author(s):  
Almog Hershko Rimon ◽  
Oded Livnah ◽  
Inna Rozman Grinberg ◽  
Lizett Ortiz de Ora ◽  
Oren Yaniv ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Unexpected Finding ◽  
Calcium Binding Site ◽  
Three Dimensional Structure ◽  
Loop Region ◽  
Carbohydrate Binding Modules ◽  
Cellulose Binding

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Å resolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

scholarly journals Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1400-1413 ◽  
Author(s):  
R Niederman ◽  
P C Amrein ◽  
J Hartwig
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Molar Ratio ◽  
Computer Assisted ◽  
Three Dimensional Structure ◽  
Actin Binding Protein ◽  
Critical Point Drying

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

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