Properties of Hybrid Active Sites in Oligomeric Proteins: Kinetic and Ligand Binding Studies with Chloramphenicol Acetyltransferase Trimers

Biochemistry ◽  
1995 ◽  
Vol 34 (19) ◽  
pp. 6416-6422 ◽  
Author(s):  
Philip J. Day ◽  
Iain A. Murray ◽  
William V. Shaw
Keyword(s):  
Ligand Binding ◽  
Active Sites ◽  
Chloramphenicol Acetyltransferase ◽  
Binding Studies ◽  
Oligomeric Proteins

scholarly journals Interbase-FRET binding assay for pre-microRNAs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mattias Bood ◽  
Anna Wypijewska del Nogal ◽  
Jesper R. Nilsson ◽  
Fredrik Edfeldt ◽  
Anders Dahlén ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Drug Targets ◽  
Structural Changes ◽  
Small Sample Size ◽  
Resonance Energy ◽  
Small Sample ◽  
Titration Calorimetry ◽  
Drug Candidate ◽  
Binding Studies ◽  
Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

The identification of cytoplasmic membrane calcium-entry channels by ligand-binding studies

1984 ◽  
Vol 12 (6) ◽  
pp. 941-943 ◽  
Author(s):  
H. GLOSSMANN ◽  
A. GOLL ◽  
D. R. FERRY ◽  
M. ROMBUSCH
Keyword(s):  
Ligand Binding ◽  
Cytoplasmic Membrane ◽  
Calcium Entry ◽  
Binding Studies

scholarly journals Comparative studies on peptides representing the so-called tachykinin-like region of the Alzheimer Aβ peptide [Aβ(25–35)]

1998 ◽  
Vol 336 (2) ◽  
pp. 419-427 ◽  
Author(s):  
Omar M. A. EL-AGNAF ◽  
G. Brent IRVINE ◽  
Geraldine FITZPATRICK ◽  
W. Kenneth GLASS ◽  
David J. S. GUTHRIE
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Comparative Studies ◽  
Aβ Peptide ◽  
Amyloid Protein ◽  
Binding Studies ◽  
Nk1 Receptors ◽  
Ligand Displacement ◽  
Β Amyloid

In an attempt to answer the question of whether or not the so-called tachykinin-like region of the Alzheimer β-amyloid protein [Aβ(25–35)] can act as a tachykinin, the sequences Aβ(25–35), Aβ(25–35)amide and their norleucine-35 and phenylalanine-31 analogues were synthesized. These peptides were examined with ligand binding studies, electron microscopy, CD and NMR. In all cases some differences were found between the Aβ(25–35) analogue and the corresponding Phe31 peptide. In addition, in ligand displacement studies on tachykinin NK1 receptors, only the Phe31 analogue showed activity comparable to that of genuine tachykinins. We conclude that peptides based on Aβ(25–35) but with a Phe residue at position 31 do display properties typical of a tachykinin, but that peptides with Ile at this position do not.

scholarly journals Identification of protein pockets and cavities by Euclidean Distance Transform

2018 ◽  
Author(s):  
Sebastian Daberdaku
Keyword(s):  
Ligand Binding ◽  
Active Sites ◽  
Euclidean Distance ◽  
Protein Structures ◽  
Ligand Docking ◽  
Distance Transform ◽  
Structure Based Drug Design ◽  
Distance Map ◽  
Euclidean Distance Transform ◽  
Protein Pockets

Protein pockets and cavities usually coincide with the active sites of biological processes, and their identification is significant since it constitutes an important step for structure-based drug design and protein-ligand docking applications. This paper presents a novel purely geometric algorithm for the detection of ligand binding protein pockets and cavities based on the Euclidean Distance Transform (EDT). The EDT can be used to compute the Solvent-Excluded surface for any given probe sphere radius value at high resolutions and in a timely manner. The algorithm is adaptive to the specific candidate ligand: it computes two voxelised protein surfaces using two different probe sphere radii depending on the shape of the candidate ligand. The pocket regions consist of the voxels located between the two surfaces, which exhibit a certain minimum depth value from the outer surface. The distance map values computed by the EDT algorithm during the second surface computation can be used to elegantly determine the depth of each candidate pocket and to rank them accordingly. Cavities on the other hand, are identified by scanning the inside of the protein for voids. The algorithm determines and outputs the best k candidate pockets and cavities, i.e. the ones that are more likely to bind to the given ligand. The method was applied to a representative set of protein-ligand complexes and their corresponding unbound protein structures to evaluate its ligand binding site prediction capabilities, and was shown to outperform most of the previously developed purely geometric pocket and cavity search methods.

Zymogen activation: a new system for homogeneous ligand-binding assay.

1984 ◽  
Vol 30 (9) ◽  
pp. 1452-1456 ◽  
Author(s):  
D A Blake ◽  
M T Skarstedt ◽  
J L Shultz ◽  
D P Wilson
Keyword(s):  
Ligand Binding ◽  
Active Sites ◽  
Binding Assay ◽  
Complete System ◽  
Coagulation Cascade ◽  
Ligand Binding Assay ◽  
Factor X ◽  
Zymogen Activation ◽  
Assay Procedure ◽  
Biotin Binding

Abstract In this ligand-binding assay procedure, sensitivity is enhanced by successive generation of enzyme active sites via two zymogens from the blood-coagulation cascade: Factor X and prothrombin. A protease fraction from Russell's viper venom (RVV) acts upon Factor X, initiating a two-step cascade that culminates in generation of thrombin, the activity of which is monitored with a chromogenic substrate. In the model presented here, the analyte of interest, biotin, is covalently coupled to Factor X. In the presence of avidin, a biotin-binding protein, RVV cannot initiate cascade activity; however, the inhibition can be competitively overcome by addition of free biotin to the reaction mixture. In a complete system, the dose-response curve is linear from 20 to 100 nmol of biotin per liter. Such an assay offers improved sensitivity over many radioisotope-independent immunoassay methods, and may be applicable to a wide variety of analytes.

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