scholarly journals Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain

1986 ◽  
Vol 238 (3) ◽  
pp. 715-719 ◽  
Author(s):  
R S Mani ◽  
C M Kay
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Difference Spectrum ◽  
Bound State ◽  
Bovine Brain ◽  
Tyrosine Residue ◽  
Difference Spectroscopy ◽  
Native Protein ◽  
Binding Properties ◽  
Helical Content

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.

scholarly journals Spectroscopic studies on Tb3+ binding to S-100a protein

1987 ◽  
Vol 244 (3) ◽  
pp. 559-563 ◽  
Author(s):  
R S Mani ◽  
C M Kay
Keyword(s):  
Conformational Changes ◽  
Binding Assay ◽  
Emission Spectra ◽  
Spectroscopic Studies ◽  
Spectral Studies ◽  
Difference Spectroscopy ◽  
Fluorescence Excitation ◽  
Fluorescence Studies ◽  
Helical Content ◽  
Direct Binding

Direct binding assay and fluorescence studies revealed that S-100a protein binds 2 mol of Tb3+/mol of protein at pH 6.6. The protein binds Tb3+ much more tightly than Ca2+, and the upper limit of the observed Kd value for Tb3+ is 3.5 × 10(-6) M. The Tb3+-binding site on the protein must be close to a tyrosine residue, as indicated by fluorescence excitation and emission spectra, where energy transfer from tyrosine is noted. Addition of Tb3+ resulted in a conformational change in the protein, as revealed by u.v.-difference spectroscopy and c.d. studies. Far-u.v. c.d. studies indicated the helical content to decrease from approx. 39% to 35% in the presence of Tb3+. From u.v.-difference-spectroscopy results the single tryptophan and the tyrosine chromophores in S-100a protein are blue-shifted (i.e. exposed to the solvent) in the presence of Tb3+ and the observed conformational changes are similar to those induced by Ca2+, suggesting that Tb3+ can be employed as a Ca2+ analogue in spectral studies with S-100a protein.

Antibody-hapten interactions in solution

1975 ◽  
Vol 272 (915) ◽  
pp. 53-74 ◽  
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Conformational Changes ◽  
Difference Spectrum ◽  
Spin Labels ◽  
Amino Acid Residues ◽  
Difference Spectroscopy ◽  
Relaxation Rates ◽  
Reciprocal Effect ◽  
Myeloma Protein

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150000, but enzymic digestion yields the Fv fragment of molecular mass 25000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pK a values of about 8.1, 6.9 and 6.1. The histidine resonances with pK a values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102 H and His 97 L . The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd hi water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd hi. At pH 5.5 there is one tight binding site for the lanthanides (K D ≈ 80 μm) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd hi quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.

scholarly journals Spectral studies on the cadmium-ion-binding properties of bovine brain S-100b protein

1991 ◽  
Vol 276 (1) ◽  
pp. 13-18 ◽  
Author(s):  
H Donato ◽  
R S Mani ◽  
C M Kay
Keyword(s):  
Absorption Band ◽  
Fluorescence Intensity ◽  
Fluorescence Emission ◽  
Bovine Brain ◽  
Spectral Studies ◽  
Tyrosine Residue ◽  
Difference Spectroscopy ◽  
Model Complexes ◽  
Emission Maximum ◽  
Fluorescence Measurements

The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.

scholarly journals The 48 kDa Ca2+-binding protein of bovine brain

1987 ◽  
Vol 246 (1) ◽  
pp. 67-74 ◽  
Author(s):  
M Tokuda ◽  
N C Khanna ◽  
D M Waisman
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Brain ◽  
Binding Properties ◽  
Tyrosine Residues ◽  
Tyrosine Protein Kinase ◽  
Stokes Radius ◽  
Phosphoamino Acid Analysis ◽  
Acidic Nature

A Ca2+-binding protein of molecular mass 48 kDa and named ‘CAB-48’ has been purified from bovine brain 100,000 g supernatant. About 30 mg of CAB-48 was purified from 1 kg of bovine brain. The protein has been characterized with respect to its physical, chemical and Ca2+-binding properties. It has an apparent molecular mass of 48 kDa by SDS/polyacrylamide-gel-electrophoresis and 75.2 kDa from sedimentation-velocity and Stokes-radius data. The acidic nature of the molecule is suggested by its pI of 4.7. In the presence of 3.0 mM-MgCl2 and 150 mM-KCl, CAB-48 binds 1.0 mol of Ca2+/mol of protein with an apparent Kd of 15 microM. A tyrosine protein kinase partially purified from rat spleen catalysed the incorporation of 0.73 mol of phosphate/mol of CAB-48, and phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues.

scholarly journals Alteration of Substrate-Induced Conformational Changes of Escherichia coli Melibiose Permease by Mutating Arg149

2020 ◽  
Author(s):  
Yibin Lin
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Structural Changes ◽  
Spectroscopic Techniques ◽  
Difference Spectroscopy ◽  
Difference Spectra ◽  
Binding Properties ◽  
Structure Changes ◽  
Β Sheets ◽  
Α Helix

AbstractFourier transform infrared difference spectroscopy and fluorescence spectroscopic techniques have been used to obtain information about substrate-induced structural changes of the melibiose permease mutant R149C, compared with the Cys-less, which were reconstituted into liposomes. ATR-FTIR evidences show that Na+-induced difference spectra of R149C and Cys-less are similar. However, Na+ induces some new peaks for R149C mutant permease. This means that replacement of Arg-149 by Cys may affect the structure of MelB, and then affect the binding of Na+. Melibiose-induced difference spectra of R149C in the presence of Na+ show some peaks in the amide I region not seen in Cys-less, corresponding to turns, β-sheets, α-helix changes. This suggests that R149C mutant permease undergo some different secondary structure changes compared to Cys-less mutant permease, when binding melibiose. Comparison of the permease intrinsic fluorescence variations of R149C and Cys-less indicate that there are similar substrate binding properties between R149C and Cys-less. When analyzing the effects of different sugars it appears that the R149C mutant is more sensitive to the sugar. All these data indicate that replacement of Arg-149 by Cys will affect Na+ and sugar binding, and enhance the selectivity and sensitivity to sugars.

scholarly journals The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Olga Ilinskaya ◽  
Vera Ulyanova ◽  
Irina Lisevich ◽  
Elena Dudkina ◽  
Nataliya Zakharchenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Concentration ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Culture Fluid ◽  
Size Exclusion ◽  
High Protein Concentration ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Hypochromic Effect

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

scholarly journals Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis

1981 ◽  
Vol 197 (1) ◽  
pp. 105-109 ◽  
Author(s):  
D R Thatcher ◽  
B Hodson
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complex Mixture ◽  
Effect Of Temperature ◽  
Electrophoretic Method ◽  
Denatured State ◽  
Double Stranded Dna ◽  
Electrophoresis Method ◽  
Dna Restriction ◽  
Irreversible Unfolding

A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun. At the end of the run the gels were stained and the effect of temperature on mobility observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton [(1979) J. Mol. Biol. 129, 253-264]. The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.

scholarly journals The C2A-C2B Linker Defines the High Affinity Ca2+ Binding Mode of Rabphilin-3A

2006 ◽  
Vol 282 (7) ◽  
pp. 5015-5025 ◽  
Author(s):  
Pierre Montaville ◽  
Christine Schlicker ◽  
Andrei Leonov ◽  
Markus Zweckstetter ◽  
George M. Sheldrick ◽  
...  
Keyword(s):  
Binding Mode ◽  
Bound State ◽  
C2 Domain ◽  
Binding Assays ◽  
Host Proteins ◽  
Binding Properties ◽  
C2 Domains ◽  
Phospholipid Binding ◽  
Acidic Residues ◽  
Structural Base

The Ca2+ binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca2+ binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca2+-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca2+ ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca2+ binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca2+-bound state of the C2B domain. In addition, this Ca2+ binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca2+ binding mode not only shows how a C2 domain increases its intrinsic Ca2+ affinity, but also provides the structural base for an atypical protein-Ca2+-phospholipid binding mode of rabphilin-3A.

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