Algorithm for the Analysis of Tryptophan Fluorescence Spectra and Their Correlation with Protein Structural Parameters

Hixon, John; Reshetnyak, Yana K.

doi:10.3390/a2031155

Cited by 29 publications

(22 citation statements)

References 81 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The maximum position at 343 nm observed for OCP (Fig. 2 A) is characteristic for Trp contacting with the structured water molecules (63). This is in good agreement with the results of x-ray crystallography (19) indicating that several water molecules (including structurally conserved) are in the immediate proximity of four Trp residues in the N-domain.…”

Section: The Intrinsic Fluorescence Of Ocp Exhibits the Photocyclesupporting

confidence: 70%

The Signaling State of Orange Carotenoid Protein

Maksimov

Shirshin

Sluchanko

et al. 2015

Biophysical Journal

112

View full text Add to dashboard Cite

Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3'-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the β-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.

show abstract

Section: The Intrinsic Fluorescence Of Ocp Exhibits the Photocyclesupporting

confidence: 70%

The Signaling State of Orange Carotenoid Protein

Maksimov

Shirshin

Sluchanko

et al. 2015

Biophysical Journal

112

View full text Add to dashboard Cite

show abstract

“…It has been well established that the intrinsic fluorescence of tryptophan is sensitive to the local environment and both fluorescence intensity and fluorescence wavelength are influenced by the extent of interactions of a tryptophan with its neighboring species (51). It is also known that if a tryptophan becomes more exposed to the surrounding hydrophilic solvent, there is a shift in the barycentric mean fluorescence wavelength (λ bcm ) towards a higher wavelength (red-shift/Stokes shift) (51,52). Conversely, if a tryptophan becomes less exposed to the polar solvent, the λ bcm undergoes a spectral shift towards a lower wavelength (blue-shift/anti-Stokes shift) (51).…”

Section: Crowder-induced Changes In Intrinsic Tryptophan Fluorescencementioning

confidence: 99%

Crowder-induced Conformational Ensemble Shift inEscherichia ColiProlyl-tRNA Synthetase

Adams

Andrews

et al. 2019

Preprint

View full text Add to dashboard Cite

The effect of macromolecular crowding on the structure and function of Escherichia coli prolyl-tRNA synthetase (Ec ProRS) has been investigated using a combined experimental and theoretical method. Ec ProRS is a multi-domain enzyme; coupled-domain dynamics is essential for efficient catalysis. To gain an insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the "soft-interactions" between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a significant shift in the conformational ensemble, which is consistent with the "soft-interactions" model of the crowding effect and explained the observed alteration in kinetic parameters. Collectively, the present study demonstrated that the effects of molecular crowding on both conformational dynamics and catalytic function, are correlated. This is the first report where molecular crowding has been found to impact the conformational ensemble in the multi-domain Ec ProRS, a member of aminoacyl-tRNA synthetase family, which is central to protein synthesis in all living cells. The present study affirmed that the effect of crowders should be considered while investigating the structure-dynamics-function relationship in modular enzymes.All crowding agents were purchased from Sigma Aldrich, except for polyethylene glycol (PEG) 8000 (Fisher Scientific). Proline (≥ 99%) was also from Sigma Aldrich. Both [γ-32 P] ATP and [ 32 P] PP i were purchased through Perkin Elmer, Shelton, CT. Overexpression and purification of Ec ProRSWild-type (WT) Ec ProRS was overexpressed in SG13009 (pREP4) competent cells using 0.1 mM isopropyl β-D-thiogalactoside for 4 hours at 37 °C. Histidine-tagged WT Ec ProRS was purified using Talon cobalt affinity resin column; 100 mM imidazole was used to elute the protein (24,25). The Bio-Rad protein assay (Bio-Rad Laboratories) was used to determine total concentration of protein. An active-site titration was performed to determine the concentration of active protein (26). Enzyme kineticsCrowding agent concentration variation. The ATP-PP i exchange assay was performed, following the protocol described elsewhere, to examine the effect of increasing concentrations of crowding agents on proline activation (eq.1) by Ec ProRS (27). In ATP-PP i exchange assay, radiolabeled PP i ( 32 PP i ) and non-radiolabeled ATP were used and the percent product (Pro-AMP) formation at 20 minutes post-initiation of the reaction was measured in the presence of crowding agents. In the present study, the amount of Pro-AMP formed was indirectly measured by monitoring the amount of 32 P-ATP formed via the reverse reaction of eq. 1 (ProRS + Pro + ATP ⇌ ProRS·(Pro-AMP) + PP i ). The percent product formation was calculated from the ratio of the Pro-A...

show abstract

“…The decreased tyrosine and tryptophan fluorescence may indicate a change in the conformation of protein molecules due to the unfolding globule amino acid residues becoming more accessible for oxidants (Giulivi et al 2003;Hixon and Reshetnyak 2009). A change in the conformation of protein molecules can lead to changes in their functional and regulatory capabilities.…”

Section: Nhmentioning

confidence: 99%

Analysis of the adhesion activity of peritoneal macrophages after exposure to radiation from a gas-discharge plasma and mercury lamp

Архипова

Пискарев²,

Иванова³

2018

gpb

View full text Add to dashboard Cite

The aim of the work was to study the influence of UV radiation of a spark discharge plasma and a mercury lamp on the state of membrane structures of peritoneal macrophages. The objects of the study were peritoneal macrophages of rats. The total number of cells after exposure and their viability were analyzed. Oxidative modification of proteins was recorded by fluorescence of tryptophan, tyrosine and products of non-enzymatic glycosylation of proteins. The concentration of sialic acids was determined spectrophotometrically, and the intensity of adhesion properties of cells was estimated by the ability to adhere to the plastic. It was shown that the radiation of a spark discharge plasma and UV lamp with the selected exposure regimes affect the structural components of membranes of peritoneal macrophages. The ability to adhere is enhanced by short exposure regimes, and under long-term conditions, adhesion properties decrease. The change in adhesion is probably associated with a decrease in the concentration of sialic acids on the cell surface, as well as with the intensification of oxidative modification of proteins. It has been established that spark plasma and UV lamp radiation promote the oxidation of aromatic amino acids and the accumulation of glycosylation products of proteins.

show abstract

Algorithm for the Analysis of Tryptophan Fluorescence Spectra and Their Correlation with Protein Structural Parameters

Cited by 29 publications

References 81 publications

The Signaling State of Orange Carotenoid Protein

The Signaling State of Orange Carotenoid Protein

Crowder-induced Conformational Ensemble Shift inEscherichia ColiProlyl-tRNA Synthetase

Analysis of the adhesion activity of peritoneal macrophages after exposure to radiation from a gas-discharge plasma and mercury lamp

Contact Info

Product

Resources

About