Distance‐dependent Fluorescence Quenching of Tryptophan by Acrylamide

Lakowicz, Joseph R.; Zelent, Bogumił; Gryczyński, Ignacy; Kuba, Józef; Johnson, Michael L.

doi:10.1111/j.1751-1097.1994.tb05092.x

Cited by 35 publications

(38 citation statements)

References 34 publications

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“…The Stern-Volmer quenching constant K D quantitatively describes this accessibility [45, 46]. The K D values for W 601 , W 669 and N-acetyl tryptophanamide were determined under native and denatured conditions using acrylamide as a quencher.…”

Section: Resultsmentioning

confidence: 99%

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

et al. 2009

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ANF-RGC$ membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques ofsteady-state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains themechanism of the model-based predicted transduction role of the ARM's structural motif, 669 WTAPELL 675 . This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the 669 WTAPELL 675 motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.

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Section: Resultsmentioning

confidence: 99%

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

et al. 2009

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“…Other studies have suggested that dynamical quenching can occur via a distance-dependent through-space interaction, 61,62 although the quencher must still approach within 3 Å of the fluorophore. 61,62 Trp87 resides >8 Å from the closest approach to bulk solvent, rendering it too far within the protein core to have random long-range interactions that distort the quenching behavior.…”

Section: Discussionmentioning

confidence: 99%

Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

Meadows

Klinman

2014

J. Phys. Chem. B

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Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context of spatially distinct differences in enthalpic barriers for protein conformational sampling that may be related to catalysis.

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“…5) In this expression b depends on quencher concentration and diffusion coefficient. Such decays can be resolved by multi-exponential models but that would be erroneous since the decays are non-exponential due to a distance-dependent occasional interaction with the quencher [39,40]. The best model to fit these non-exponential decays with a single exponent is a lifetime distribution model.…”

Section: Effect Of Fluorescence Quenching On 2ap Lifetime Distributionmentioning

confidence: 99%

“…The best model to fit these non-exponential decays with a single exponent is a lifetime distribution model. We used acrylamide to quench 2AP fluorescence in a highly viscous solution (80:20 glycerol:water mixture) to detect the transient effect clearly [39,40]. One molar acrylamide quenches 2AP fluorescence by 50% with no detectable spectral shift ( Figure 4).…”

Section: Effect Of Fluorescence Quenching On 2ap Lifetime Distributionmentioning

confidence: 99%

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

Bharill

Sarkar

Ballin

et al. 2008

Analytical Biochemistry

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The fluorescent adenine analogue 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV/Vis spectroscopy and circular dichroism. However, while the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multi-exponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we have tested the utility of multi-exponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities, or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case, except folded RNA which required a bimodal distribution, 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties, which will further the utility of this Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

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Distance‐dependent Fluorescence Quenching of Tryptophan by Acrylamide

Cited by 35 publications

References 34 publications

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

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