scholarly journals Cucurbitacin Δ23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions

1986 ◽  
Vol 233 (3) ◽  
pp. 649-653 ◽  
Author(s):  
H W Dirr ◽  
J C Schabort ◽  
C Weitz
Keyword(s):  
Fluorescence Quenching ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Dissociation Constants ◽  
Protein Molecule ◽  
Cucurbita Maxima ◽  
Protein Fluorescence ◽  
Tryptophan Residues ◽  
Ligand Interactions ◽  
Ph Range

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 × 10(-7) cm2 × s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

scholarly journals Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes

1997 ◽  
Vol 321 (2) ◽  
pp. 355-360 ◽  
Author(s):  
Shashiprabha DASGUPTA ◽  
Dipak DASGUPTA ◽  
Aruna CHATTERJEE ◽  
Susweta BISWAS ◽  
Birendra B. BISWAS
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Dissociation Constants ◽  
Spectroscopic Studies ◽  
Binding Affinities ◽  
Inositol Trisphosphate Receptor ◽  
Single Temperature ◽  
Tryptophan Residues ◽  
Fluorescence Quencher ◽  
Trisphosphate Receptor

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3 and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro.Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 ŶC), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 ƁM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores, because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500ŷ200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting SternŐVolmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205Ő250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG), for the formation of the two InsP3Őreceptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.

scholarly journals Binding Studies of AICAR and Human Serum Albumin by Spectroscopic, Theoretical, and Computational Methodologies

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5410
Author(s):  
Shokoufeh Hashempour ◽  
Nahid Shahabadi ◽  
Aishat Adewoye ◽  
Brennen Murphy ◽  
Camaray Rouse ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Fluorescence Quenching ◽  
Serum Albumin ◽  
Human Serum ◽  
Binding Site ◽  
Conformational Changes ◽  
Flufenamic Acid ◽  
Drug Candidate ◽  
Protein Fluorescence ◽  
Binding Studies

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR’s pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR–HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern–Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR–HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA’s binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR–HSA complexes, reaffirming that AICAR binds to both site I and site II.

scholarly journals Effect of pH on the Hydrolytic Kinetics of Gamma-Glutamyl Transferase fromBacillus subtilis

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Sharath Balakrishna ◽  
Asmita Prabhune
Keyword(s):  
Conformational Changes ◽  
Quaternary Structure ◽  
Gamma Glutamyl Transferase ◽  
Effect Of Ph ◽  
Gamma Glutamyl ◽  
Steady State Kinetics ◽  
Kinetic Transformation ◽  
Ph Range ◽  
Glutamyl Transferase ◽  
Kinetics Of

The effect of pH on the steady state kinetics of gamma-glutamyl transferase (GGT) fromBacillus subtiliswas examined using glutamyl-(3-carboxyl)-4-nitroanilide as the chromogenic reporter substrate. The enzyme was active in the pH range 7.0–11.0 with the optimum activity at pH 11.0. We noticed a pH dependent transformation in the nature of substrate consumption kinetics. The substrate saturation curves were hyperbolic in the pH range 7.0–9.0 but changed into sigmoid form at pH 10.0 and 11.0. Hill’s coefficients were >1. We also analysed the effect of pH on the structure of the enzyme. The circular dichroism spectra of the enzyme sample at pH 9.0 and 11.0 were coincidental in both far and near UV regions indicating conservation of the secondary and tertiary structures, respectively. The molecular weight of the enzyme sample was the same in both pH 7.0 and 11.0 indicating conservation of the quaternary structure. These results show that the kinetic transformation does not involve significant conformational changes. Cooperative binding of multiple substrate molecules may not be the basis for the sigmoid kinetics as only one substrate binding site has been noticed in the reported crystal structures ofB. subtilisGGT.

Fluorescence study on transmembrane Ca2+ gradient-mediated conformation changes of sarcoplasmic reticulum Ca2+-ATPase

1994 ◽  
Vol 14 (6) ◽  
pp. 309-317 ◽  
Author(s):  
Y. P. Tu ◽  
F. Y. Yang
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Fluorescence Quenching ◽  
Conformational Changes ◽  
Dynamic Change ◽  
Parasitic Fungus ◽  
Time Resolved Fluorescence ◽  
Fluorescence Study ◽  
Time Resolved ◽  
Fluorescence Measurements ◽  
Tryptophan Residues

The conformational states of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles with or without a thousand-fold transmembrane Ca2+ gradient have been studied by fluorescence spectroscopy and fluorescence quenching. In consequence of the establishment of the transmembrane Ca2+ gradient, the steady-state fluorescence results revealed a reproducible 8% decrease in the intrinsic fluorescence while time-resolved fluorescence measurements showed that 13 tryptophan residues in SR · Ca2+-ATPase could be divided into three groups. The fluorescence lifetime of one of these groups increased from 5.5 ns to 5.95 ns in the presence of a Ca2+ gradient. Using KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus, growing in Yunnan, China), the fluorescence quenching further indicated that the dynamic change of this tryptophan group, located at the protein-lipid interface, is a characteristic of transmembrane Ca2+ gradient-mediated conformational changes in SR · Ca2+-ATPase.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

2019 ◽  
Vol 26 (10) ◽  
pp. 743-750 ◽  
Author(s):  
Remya Radha ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Kinetic Studies ◽  
Structural Features ◽  
Structure Stability ◽  
Kcal Mole ◽  
Scanning Calorimetry ◽  
Wide Range ◽  
Ph Dependent ◽  
Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

19F-NMR in Target-based Drug Discovery

2019 ◽  
Vol 26 (26) ◽  
pp. 4964-4983 ◽  
Author(s):  
CongBao Kang
Keyword(s):  
Drug Discovery ◽  
Conformational Changes ◽  
Protein Structures ◽  
19F Nmr ◽  
Binding Affinities ◽  
Protein Ligand Interactions ◽  
Compound Libraries ◽  
Ligand Interactions ◽  
Reference Ligand ◽  
Hit Identification

Solution NMR spectroscopy plays important roles in understanding protein structures, dynamics and protein-protein/ligand interactions. In a target-based drug discovery project, NMR can serve an important function in hit identification and lead optimization. Fluorine is a valuable probe for evaluating protein conformational changes and protein-ligand interactions. Accumulated studies demonstrate that 19F-NMR can play important roles in fragment- based drug discovery (FBDD) and probing protein-ligand interactions. This review summarizes the application of 19F-NMR in understanding protein-ligand interactions and drug discovery. Several examples are included to show the roles of 19F-NMR in confirming identified hits/leads in the drug discovery process. In addition to identifying hits from fluorinecontaining compound libraries, 19F-NMR will play an important role in drug discovery by providing a fast and robust way in novel hit identification. This technique can be used for ranking compounds with different binding affinities and is particularly useful for screening competitive compounds when a reference ligand is available.

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