scholarly journals Depolarization of the intrinsic and extrinsic fluorescence of pepsinogen and pepsin

1970 ◽  
Vol 116 (3) ◽  
pp. 341-348 ◽  
Author(s):  
F. W. J. Teale ◽  
R. A. Badley
Keyword(s):  
Relaxation Times ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Molecule ◽  
Rotational Relaxation ◽  
Collisional Quenching ◽  
Shape Factors ◽  
Emission Anisotropy ◽  
Specific Orientation ◽  
Tryptophan Emission

1. The effects on the intrinsic tryptophan emission anisotropy of pepsin and pepsinogen solutions produced by (a) changes in temperature, (b) increases in viscosity with added glycerol at constant temperature and (c) decreases in lifetime through collisional quenching by potassium iodide were measured at several excitation wavelengths. The rotational-relaxation times calculated from results provided by method (b) approximate to the theoretical values for the two proteins, on taking hydration and shape factors into account, on the basis of random orientation of the tryptophan groups within the macromolecules. Differences between the results provided by methods (b) and (c) are attributable to inter-tryptophan resonance-energy-transfer depolarization, and the anomalous values recorded in method (a) can be attributed to the temperature-dependence of the limiting anisotropies. 2. Two different monomeric conjugates of pepsin, each containing one extrinsic fluorescent group per macromolecule, gave widely different relaxation times. This difference may arise from a specific orientation of the emission dipole in the enzyme. In active-site-labelled pepsin (1-dimethylaminonaphthalene-5-sulphonylphenylalanine–pepsin) this orientation would be approximately parallel to the symmetry axis of the equivalent ellipsoid, whereas in the other conjugate (1-dimethylaminonaphthalene-5-sulphonyl-pepsin) the orientation may be roughly normal to this direction, or some independent rotation of parts of the protein molecule is possible.

scholarly journals RET-enhanced measurement of relaxation time constants of LOV2-based optogenetic actuators

2020 ◽  
Author(s):  
Li-Li Li ◽  
Michael J Courtney
Keyword(s):  
Fluorescent Protein ◽  
Relaxation Times ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alternative Methods ◽  
Relaxation Rates ◽  
Experimental Conditions ◽  
Whole Cell Lysate ◽  
Activated State ◽  
Relaxation Time Constants

Abstract Optogenetic actuators exist in either active or inactive states. Absorption of light drives transition of the chromophore to the activated state, whereas thermal processes typically cause gradual relaxation to the initial or dark state. Relaxation rates determine how often activation light needs to be applied to maintain the activated state, but this rate is strongly affected by temperature and sequences surrounding the photosensor domain. Application of existing cellular optogenetic actuators and optimization of new ones therefore requires knowledge of the relaxation rates under the experimental conditions in which they are used. When proteins targeted by the actuator do not generate immediately visible responses, alternative methods are required to determine relaxation times. We describe a simple yet sensitive procedure to measure the relaxation rate constant for an optogenetic actuator. By using resonance energy transfer with a fused fluorescent protein tag to detect the change in chromophore state, low amounts of whole cell lysate are sufficient to perform the measurement.

Probing native metal ion association sites through quenching of fluorophores in the nucleotide-binding domains of the ABC transporter MsbA

2017 ◽  
Vol 474 (12) ◽  
pp. 1993-2007 ◽  
Author(s):  
Daiki Tatsumi ◽  
Kei Nanatani ◽  
Yuto Koike ◽  
Kiyoto Kamagata ◽  
Satoshi Takahashi ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Conformational Changes ◽  
Metal Ion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ion Association ◽  
Nucleotide Binding ◽  
Small Scale ◽  
Collisional Quenching ◽  
Binding Domains

ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from Escherichia coli, with a fluorescent probe, Alexa532, within the NBDs. ATP application elicited collisional quenching, whereas no quenching was observed after the addition of ATP analogs or ATP hydrolysis inhibitors. The Alexa532-conjugated MsbA variants exhibited transition metal ion Förster resonance energy transfer (tmFRET) after the addition of Ni2+, and ATP decreased this Ni2+-mediated FRET of the NBDs. Structure modeling developed from crystallographic data and examination of tmFRET measurements of MsbA variants in the absence of ATP revealed the presence of metal ion-associated pockets (MiAPs) in the NBDs. Three histidines were predicted to participate in chelating Ni2+ in the two possible MiAPs. Performing histidine-substitution experiments with the NBDs showed that the dissociation constant for Ni2+ of MiAP2 was smaller than that of MiAP1. The structural allocation of the MiAPs was further supported by showing that the addition of Cu2+ resulted in higher quenching than Ni2+. Taken together, the present study showed that the NBDs contain two native binding sites for metal ions and ATP addition affects the Ni2+-binding activity of the MiAPs.

scholarly journals Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

1979 ◽  
Vol 27 (1) ◽  
pp. 56-64 ◽  
Author(s):  
S S Chan ◽  
D J Arndt-Jovin ◽  
T M Jovin
Keyword(s):  
Energy Transfer ◽  
Flow System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
Emission Anisotropy ◽  
Lectin Receptors ◽  
Surface Receptors ◽  
Erythroleukemia Cells ◽  
Donor Acceptor

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.

scholarly journals RET-enhanced measurement of relaxation time constants of LOV2-based optogenetic actuators

2020 ◽  
Author(s):  
Li-Li Li ◽  
Michael J Courtney
Keyword(s):  
Fluorescent Protein ◽  
Relaxation Times ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alternative Methods ◽  
Relaxation Rates ◽  
Experimental Conditions ◽  
Whole Cell Lysate ◽  
Activated State ◽  
Relaxation Time Constants

Abstract Optogenetic actuators exist in either active or inactive states. Absorption of light drives transition of the chromophore to the activated state, whereas thermal processes typically cause gradual relaxation to the initial or dark state. Relaxation rates determine how often activation light needs to be applied to maintain the activated state, but this rate is strongly affected by temperature and sequences surrounding the photosensor domain. Application of existing cellular optogenetic actuators and optimization of new ones therefore requires knowledge of the relaxation rates under the experimental conditions in which they are used. When proteins targeted by the actuator do not generate immediately visible responses, alternative methods are required to determine relaxation times. We describe a simple yet sensitive procedure to measure the relaxation rate constant for an optogenetic actuator. By using resonance energy transfer with a fused fluorescent protein tag to detect the change in chromophore state, low amounts of whole cell lysate are sufficient to perform the measurement.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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