Protein Fluorescence (protein + fluorescence)

Distribution by Scientific Domains


Selected Abstracts


Recombinant human glucose-6-phosphate dehydrogenase

FEBS JOURNAL, Issue 14 2002
Evidence for a rapid-equilibrium random-order mechanism
Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver,Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 µm, close to the value of 6.8 µm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding. [source]


A comparison of the urea-induced unfolding of apoflavodoxin and flavodoxin from Desulfovibrio vulgaris

FEBS JOURNAL, Issue 1 2002
Brian Ó Nuallain
The kinetics and thermodynamics of the urea-induced unfolding of flavodoxin and apoflavodoxin from Desulfovibrio vulgaris were investigated by measuring changes in flavin and protein fluorescence. The reaction of urea with flavodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s,1 in 3 m urea in 25 mm sodium phosphate at 25 °C), and it results in the dissociation of FMN. The rate of unfolding of apoflavodoxin depends on the urea concentration, while the reaction with the holoprotein is independent of urea. The rates decrease in high salt with the greater effect occurring with apoprotein. The fluorescence changes fit two-state models for unfolding, but they do not exclude the possibility of intermediates. Calculation suggests that 21% and 30% of the amino-acid side chains become exposed to solvent during unfolding of flavodoxin and apoflavodoxin, respectively. The equilibrium unfolding curves move to greater concentrations of urea with increase of ionic strength. This effect is larger with phosphate than with chloride, and with apoflavodoxin than with flavodoxin. In low salt the conformational stability of the holoprotein is greater than that of apoflavodoxin, but in high salt the relative stabilities are reversed. It is calculated that two ions are released during unfolding of the apoprotein. It is concluded that the urea-dependent unfolding of flavodoxin from D. vulgaris occurs because apoprotein in equilibrium with FMN and holoprotein unfolds and shifts the equilibrium so that flavodoxin dissociates. Small changes in flavin fluorescence occur at low concentrations of urea and these may reflect binding of urea to the holoprotein. [source]


Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

JOURNAL OF NEUROCHEMISTRY, Issue 2 2007
Sannah Zoffmann
Abstract Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 Å for the fluorophore linked to positions 1,10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain. [source]


On Spectral Relaxation in Proteins,,§

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2000
Joseph R. Lakowicz
ABSTRACT During the past several years there has been debate about the origins of nonexponential intensity decays of intrinsic tryptophan (trp) fluorescence of proteins, especially for single tryptophan proteins (STP). In this review we summarize the data from diverse sources suggesting that time-dependent spectral relaxation is a ubiquitous feature of protein fluorescence. For most proteins, the observations from numerous laboratories have shown that for trp residues in proteins (1) the mean decay times increase with increasing observation wavelength; (2) decay associated spectra generally show longer decay times for the longer wavelength components; and (3) collisional quenching of proteins usually results in emission spectral shifts to shorter wavelengths. Additional evidence for spectral relaxation comes from the time-resolved emission spectra that usually shows time-dependent shifts to longer wavelengths. These overall observations are consistent with spectral relaxation in proteins occurring on a subnanosecond timescale. These results suggest that spectral relaxation is a significant if not dominant source of nonexponential decay in STP, and should be considered in any interpretation of nonexponential decay of intrinsic protein fluorescence. [source]


Monitoring the fractionation of a whey protein isolate during dead-end membrane filtration using fluorescence and chemometric methods

BIOTECHNOLOGY PROGRESS, Issue 1 2010
Rand Elshereef
Abstract During membrane-based separation of proteins, changes in protein concentration of the permeate and retentate streams occurs over time. The current work proposes a new approach for monitoring the changes in concentrations of proteins in both permeate and retentate by making use of data collected using fluorescence spectroscopy and intrinsic protein fluorescence analyzed by multivariate statistical techniques. Whey protein isolate consists mainly of ,-lactalbumin (,-LA), ,-lactoglobulin (,-LG), and small proportion of bovine serum albumin (BSA) and was used as a model system in this study. A fiber optic probe (FOP) was used to acquire multiwavelength fluorescence spectra for permeate and retentate streams at different times during UF-based separation of the components from a multicomponent solution. Multivariate regression models were developed for predicting the concentrations of ,-LA, ,-LG, and BSA by establishing a calibration model between data acquired using the FOP and the corresponding protein concentration levels measured by size-exclusion chromatography. The model was validated using FOP data that were not previously used for calibration of the regression models. This comparison showed that concentrations of ,-LA, ,-LG, and BSA could be predicted directly from FOP data within reasonable accuracy by making use of multivariate calibration tools. This approach has several attractive features including that it is nondestructive, fast, and relatively simple to perform. This technique has potential practical applications as it could offer the opportunity for in situ monitoring of membrane filtration processes by tracking individual protein transmission and selectivity of fractionation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]