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.

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 An efficient method for FITC labelling of proteins using tandem affinity purification

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Lalith K. Chaganti ◽  
Navneet Venkatakrishnan ◽  
Kakoli Bose
Keyword(s):  
Conformational Changes ◽  
Affinity Purification ◽  
Spectroscopic Studies ◽  
Tandem Affinity Purification ◽  
Target Protein ◽  
High Quantum Efficiency ◽  
High Background ◽  
Fluorescence Studies ◽  
Background Fluorescence ◽  
Experimental Approaches

Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.

scholarly journals Comparative structural and optical spectroscopic studies of Nd3+ ion doped LaF3 and their core/shell nanoparticles

2018 ◽  
Vol 12 (1) ◽  
pp. 78-85
Author(s):  
Anees Ansari ◽  
Joselito Labis ◽  
Mohamed Manthrammel
Keyword(s):  
Surface Coating ◽  
Silica Surface ◽  
Uv Light ◽  
Emission Spectra ◽  
Spectroscopic Studies ◽  
Hexagonal Structure ◽  
Spectral Studies ◽  
Core Shell ◽  
Shell Nanoparticles ◽  
Average Grain Size

LaF3 :Nd3+ (core), LaF3 :Nd3+@LaF3 (core/shell) and LaF3 :Nd3+@LaF3@SiO2 (core/shell/SiO2) nanoparticles (NPs) were designed and synthesized at low temperature. The structure and morphology of the as-prepared nanoproducts were characterized by X-ray diffraction and transmission electron microscopy (TEM) techniques. Thermal analysis and FTIR spectral studies were conducted to examine the surface properties of the nanomaterials. The as-prepared LaF3 :Nd3+ NPs exhibited hexagonal structure and were composed of monodispersed irregularly and spherically shaped NPs with average grain size of 21 nm. TEM image showed the successful silica surface coating, which was verified by FTIR spectral analysis. The emission spectra of LaF3 :Nd3+ NPs was obtained by monitoring the emission of Nd3+ at 325 nm (Cd laser) where it exhibited the characteristic 4f?4f transitions lines originating from the Nd 4f3 configuration. Under UV light irradiation, the emission spectra revealed various strong emission transitions which were greatly affected by the surrounding silica surface coating. These observed results suggested future applications in biolabeling and light-emitting diode.

scholarly journals Harnessing intrinsic fluorescence for typing of secondary structures of DNA

2020 ◽  
Author(s):  
Michela Zuffo ◽  
Aurélie Gandolfini ◽  
Brahim Heddi ◽  
Anton Granzhan
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
Emission Spectra ◽  
Principal Component ◽  
Secondary Structures ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Linear Discriminant ◽  
Effective Manner

ABSTRACTDNA is polymorphic since, despite its ubiquitous presence as a double-stranded helix, it is able to fold into a plethora of other secondary structures both in vitro and in cells. Despite the considerable advances that have been made in understanding this structural diversity, its high-throughput investigation still faces severe limitations. This mainly stems from the lack of suitable label-free methods, combining a fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of the suitability of this phenomenon for tracking the conformational changes of DNA, we examined the intrinsic steady-state emission spectra of an 89-membered set of synthetic oligonucleotides with reported conformation (G-quadruplexes, i-motifs, single- and double stranded DNA) by means of multivariate analysis. Specifically, principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, albeit without discrimination between single- and double-stranded structures. Linear discriminant analysis of the same training set was exploited for the assessment of new sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labelling agent or dye, avoiding the related intrinsic bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T), the most fluorescent G4 structure reported to date. This property is likely to arise from the similar base-stacking geometry in both types of structures.

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol

2006 ◽  
Vol 84 (2) ◽  
pp. 126-134 ◽  
Author(s):  
Fouzia Rashid ◽  
Sandeep Sharma ◽  
M A Baig ◽  
Bilqees Bano
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Structural Features ◽  
Cd Spectroscopy ◽  
Native State ◽  
Molten Globule State ◽  
Fluorescence Measurements ◽  
Helical Content ◽  
Human Placental Cystatin

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of α-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.Key words: human placental cystatin, molten globule, acid-induced state, trifluoroethanol, methanol, CD spectroscopy, ANS fluorescence, pH, protein folding.

Raman Spectroscopic Study of Ba2+-Surfactant Interactions in Aqueous Monodisperse Alkylpolyoxyethylene Surfactant Solutions

1993 ◽  
Vol 47 (11) ◽  
pp. 1784-1787
Author(s):  
Diana C. W. Siew ◽  
Ralph P. Cooney ◽  
Michael J. Taylor
Keyword(s):  
Conformational Changes ◽  
Influencing Factor ◽  
Difference Spectroscopy ◽  
Raman Spectroscopic ◽  
Chain Component ◽  
Raman Spectroscopic Study ◽  
Surfactant Solutions ◽  
Surfactant Complex ◽  
Surfactant Interactions ◽  
The Stability

Aqueous monodisperse alkylpolyoxyethylene surfactant-Ba2+ systems were investigated to clarify the coordination effects between the polyether chain and the cation which have been previously identified in polydisperse alkylphenoxy- and alkypolyoxyethylene surfactant systems. The coordination effects are simplified in the present study due to the presence of only a single polyether chain component. Formation of the surfactant complex resulted in conformational changes of the polyether chain which were monitored by Raman difference spectroscopy and curve fitting. The present studies confirm that surfactant-complex formation is dependent upon the polyether chain adopting the TGT-TĜT conformation and that the stability of the complex increases with the number of filled chain sites. The length of the polyether chain is also an influencing factor.

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