On the interpretation of decay-associated fluorescence spectra in proteins
Calculating decay-associated spectra (DAS) is a common mode of analyzing complex fluorescence decay in proteins. Unfortunately, these spectra are attributed almost exclusively to certain species in the population of macromolecules (retainers), often without sufficient evidence. An alternative explan...
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nasplib_isofts_kiev_ua-123456789-1552282025-02-09T09:43:42Z On the interpretation of decay-associated fluorescence spectra in proteins До тлумачення спектрів, асоційованих із затуханням флюоресценції в білках К интерпретации спектров, ассоциированных с затуханием флюоресценции в белках Ladokhin, A.S. Структура та функції біополімерів Calculating decay-associated spectra (DAS) is a common mode of analyzing complex fluorescence decay in proteins. Unfortunately, these spectra are attributed almost exclusively to certain species in the population of macromolecules (retainers), often without sufficient evidence. An alternative explanation of decay heterogeneity, based on structural relaxation., was believed to require a negative DAS component. Here we demonstrate that dipolar relaxation of the indole molecule, lacking any rotameric forms, doesn't always result in negative pre-exponential amplitude. We conclude that the shape of DAS alone is not sufficient to differentiate among various causes of heterogeneity of tryptophan fluorescence decay in proteins. Расчет спектров, ассоциированных с затуханием (САЗ), является обычным путем анализа сложного затухания флюоресценции в белках. К сожалению, эти спектры почти исключительно относят к определенным видам в популяции молекул (ротамеров), часто без достаточных оснований Считалось, что альтернативное объяснение гетерогенности затухания, связанное со структурной релаксацией, требует отрицательной компоненты САЗ. В данной работе мы демонстрируем, что дипольная релаксация молекулы индола, лишенной каких-либо ротамерных форм, не всегда приводит к отрицательным при-экспоненциальным амплитудам. Мы делаем вывод о том, что форма САЗ сама по себе недостаточна, чтобы определить среди других причину гетерогенности затухания триптофановой флюоресценции в белках. Обрахунок спектрів, асоційованих із затуханням (САЗ), є звичайним шляхом аналізу складного затухання флюоресценції в білках. На жаль, ці спектри майже виключно відносять до певних видів у популяції молекул (ротамерів), часто без достатніх на те підстав. Вважалося, що альтернативне пояснення гетерогенності затухання, пов'язане зі структурною релаксацією, потребує негативної компоненти САЗ. У цій роботі ми демонструємо, що дипольна релаксація молекули індолу, позбавленої будь-яких ротамерних форм, не завжди призводить до негативних при-експоненціальних амплітуд. Ми робимо висновок стосовно того, що форма САЗ сама по собі не є достатньою, щоб вирізнити серед інших причину гетерогенності затухання триптофанової флюоресценції в білках. 2001 Article On the interpretation of decay-associated fluorescence spectra in proteins / A.S. Ladokhin // Біополімери і клітина. — 2001. — Т. 17, № 3. — С. 221-224. — Бібліогр.: 14 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.0005AE https://nasplib.isofts.kiev.ua/handle/123456789/155228 577.32 + 577.322 en Біополімери і клітина application/pdf Інститут молекулярної біології і генетики НАН України |
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Структура та функції біополімерів Структура та функції біополімерів |
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Структура та функції біополімерів Структура та функції біополімерів Ladokhin, A.S. On the interpretation of decay-associated fluorescence spectra in proteins Біополімери і клітина |
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Calculating decay-associated spectra (DAS) is a common mode of analyzing complex fluorescence decay in proteins. Unfortunately, these spectra are attributed almost exclusively to certain species in the population of macromolecules (retainers), often without sufficient evidence. An alternative explanation of decay heterogeneity, based on structural relaxation., was believed to require a negative DAS component. Here we demonstrate that dipolar relaxation of the indole molecule, lacking any rotameric forms, doesn't always result in negative pre-exponential amplitude. We conclude that the shape of DAS alone is not sufficient to differentiate among various causes of heterogeneity of tryptophan fluorescence decay in proteins. |
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Article |
| author |
Ladokhin, A.S. |
| author_facet |
Ladokhin, A.S. |
| author_sort |
Ladokhin, A.S. |
| title |
On the interpretation of decay-associated fluorescence spectra in proteins |
| title_short |
On the interpretation of decay-associated fluorescence spectra in proteins |
| title_full |
On the interpretation of decay-associated fluorescence spectra in proteins |
| title_fullStr |
On the interpretation of decay-associated fluorescence spectra in proteins |
| title_full_unstemmed |
On the interpretation of decay-associated fluorescence spectra in proteins |
| title_sort |
on the interpretation of decay-associated fluorescence spectra in proteins |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2001 |
| topic_facet |
Структура та функції біополімерів |
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https://nasplib.isofts.kiev.ua/handle/123456789/155228 |
| citation_txt |
On the interpretation of decay-associated fluorescence spectra in proteins / A.S. Ladokhin // Біополімери і клітина. — 2001. — Т. 17, № 3. — С. 221-224. — Бібліогр.: 14 назв. — англ. |
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Біополімери і клітина |
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2025-11-25T10:29:01Z |
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2025-11-25T10:29:01Z |
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1849757839700000768 |
| fulltext |
ISSN 0233-7657. Біополімери і клітина. 2001. Т. 17. № З
On the interpretation of decay-associated
fluorescence spectra in proteins
A. S. Ladokhin
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150 Academician Zabolotnoho vul., Kyiv, 03143, Ukraine
Department of Physiology and Biophysics University of California
Irvine, CA 92697-4560 U.S.A.
E. mail: ladokhin@uci.edu
Calculating decay-associated spectra (DAS) is a common mode of analyzing complex fluorescence decay
in proteins. Unfortunatelyt these spectra are attributed almost exclusively to certain species in the
population of macromolecules (rotamers), often without sufficient evidence. An alternative explanation of
decay heterogeneity, based on structural relaxation, was believed to require a negative DAS component.
Here we demonstrate that dipolar relaxation of the indole molecule, lacking any rotameric forms, doesn't
always result in negative pre-exponential amplitude. We conclude that the shape of DAS alone is not
sufficient to differentiate among various causes of heterogeneity of tryptophan fluorescence decay in
proteins.
«lt is very difficult to find a black cat in a dark room ... Especialy if its not there.»
Attributed to Confucius
Tryptophan fluorescence in proteins is known to
exhibit a complex decay, which nevertheless can be
fitted to a sum of exponentials: I it) e expC-t/rj).
The origin of the decay heterogeneity has been the
subject of multiple studies as well as the recent e-mail
discussion summarized by Dr. Callis at the meeting of
the Biological Fluorescence subgroup of the Bio
physical Society [1 ]. The two main interpretations are
the excitation-state relaxation hypothesis (reviewed in
[2, 3 ]) and the rotamer hypothesis [4—6 ]. The latter
provides a rationale for global analysis of multiple
kinetics [7]. Normally, fluorescence lifetimes, r,, are
designated to be global (i. e. shared between the data
sets), while pre-exponential amplitudes, ai9 are de
signated non-global. In this way, the global analysis
can be used to recover the spectral distribution of at
(the so-called decay-associated spectra or DAS). Ho
wever, from the very start, the authors of the Global
© A. S. LADOKHIN, 2001
approach clearly stated the complexity of interpreting
DAS when excited-state reactions are involved, and
stressed the necessity of distinguishing DAS from
species-associated spectra (SAS) [8, 9] . Unfortuna
tely, in the vast majority of the studies of tryptophan
fluorescence in proteins in which DAS are recovered,
the latter are attributed exclusively to certain species
in the population of macromolecules (rotamers).
Numerous studies indicate that chemical he
terogeneity [10] and dipolar relaxation [11, 12] in
indole's environment lead to complex fluorescence
decay. Obviously, indole is deprived of any rotameric
forms which simplifies the interpretation. The main
argument used against the relaxation hypothesis for
protein fluorescence decay heterogeneity was the
absence of the negative pre-exponential component on
the red wing of emission [1 ], thought to be a
necessary hallmark of the relaxation. Indeed, this
feature, reported for indole in glycerol rich (90 % or
more) solutions [11, 12], is seldom seen in proteins.
However the DAS that exhibits a negative component
221
mailto:ladokhin@uci.edu
LADOKHIN A. S.
Fig 1. DAS of indole in 40 % glycerol at 10 °С at two excitations indicated on the graph. Two components (symbols) represent a heterogeneity
of decay caused by dipolar relaxation. Red-edge excitation (right-hand panel) results in photo-selection of solvates which are closer to
equilibrium in the excited state, resulting in shorter second lifetime
Fig 2. DAS of tryptophan zwitterion in water and in 40 % glycerol at 10 °С measured with the 295 nm excitation. Two components (symbols)
in the case of tryptophan in water (left-hand panel) are related to rotameric forms. The three components (symbols) observed in 40 %
glycerol (right-hand panel) are similar to those observed with many different proteins. There is no possibility to determine from the appearance
of the spectra which of them are related to the excited state relaxation and which to the ground state heterogeneity
222
ON THE INTERPRETATION OF FLUORESCENCE SPECTRA IN PROTEINS
in model solutions corresponds to the shortest of three
lifetimes and might not always be observed. We had
suggested that the existence of DAS with negative
amplitude is a sufficient but not necessary indication
of excited state reactions [11 ]. Here we examine this
issue in more detail, using water/glycerol mixtures
with lower glycerol content (3/2 volume units), re
ferred to below as 40 % glycerol. All other experi
mental details and procedures for calculating DAS
were exactly the same as those used in the previous
study [11 ]. Global %2 for fits presented here were 1.1
or less.
The DAS for indole in 40 % glycerol collected at
10 °С using two different excitation wavelength are
presented in Fig. 1. For this mildly viscous solution
only two decay components are required to fit the
data and no negative preexponential is observed. Note
that the decay time of a minor component decreases
under conditions of red-edge excitation (right panel).
This change is not consistent with the two-state
relaxation in solvate (defined as a chromophore and
its solvent shell). Instead, a continuous relaxation in
the ensemble of sub-states should be considered. This
type of relaxation is well documented for organic dyes
in viscous isotropic media and when bound to lipid
vesicles (reviewed in [13]). In all cases listed above,
non-exponential behavior of fluorescence decay could
be explained using a concept of inhomogeneous bro
adening of electronic spectra of single molecular
species. There is no reason why the same concept
should not be applied to an indole chromophore in a
complex and heterogeneous environment inside the
protein.
The DAS for tryptophan zwitterion in water and
in 40 % glycerol is presented in Fig. 2. Two
components observed in water (left-hand panel) are
related to rotameric forms [4]. Addition of the
relaxation expected for the mixture (right-hand pa
nel) complicates the picture. While the longest lifetime
appears unchanged, the second one increases by a
factor of two. and the third subnanosecond component
appears. It is likely that the latter is the product of
relaxation (compare to the shortest lifetime on Fig.
1), however it might be not the only one. Overall the
three DAS obtained for this model system are very
typical for those measured with real proteins. Thus,
even in the absence of negative preexponential, the
DAS for protein could still be influenced by the
relaxation phenomena. Indiscriminate application of
the global analysis and one-sided interpretation of the
results can lead to the over interpretation of fluo
rescence data. For tryptophan fluorescence we recom
mend two recent studies [11, 14] that suggest the
experimental criteria for choosing among various
interpretations. One should be careful to chose the
physically relevant model for doing global analysis,
otherwise one risks ending up chasing a black cat in
a dark room.
Acknowledgement: Supported by W. Keck Foun
dation and GM-46823 from the NIH.
О. С Ладохін
До тлумачення спектрів, асоційованих із затуханням
флюоресценції в білках
Резюме
Обрахунок спектрів, асоційованих із затуханням (САЗ), є
звичайним шляхом аналізу складного затухання флюоресценції
в білках. На жаль, ці спектри майже виключно відносять до
певних видів у популяції молекул (ротамерів), часто без
достатніх на те підстав. Вважалося, що альтернативне
пояснення гетерогенності затухання, пов'язане зі структур
ною релаксацією, потребує негативної компоненти САЗ. У цій
роботі ми демонструємо, що дипольна релаксація молекули
індолу, позбавленої будь-яких ротамерних форм, не завжди
призводить до негативних при-експоненціальних амплітуд. Ми
робимо висновок стосовно того, що форма САЗ сама по собі
не є достатньою, щоб вирізнити серед інших причину гетеро
генності затухання триптофанової флюоресценції в білках.
А. С. Ладохин
К интерпретации спектров, ассоциированных с затуханием
флюоресценции в белках
Резюме
Расчет спектров, ассоциированных с затуханием (САЗ), явля
ется обычным путем анализа сложного затухания флюорес
ценции в белках. К сожалению, эти спектры почти исключи
тельно относят к определенным видам в популяции молекул
(ротамеров), часто без достаточных оснований Считалось,
что альтернативное объяснение гетерогенности затухания,
связанное со структурной релаксацией, требует отрицатель
ной компоненты САЗ. В данной работе мы демонстрируем,
что дипольная релаксация молекулы индола, лишенной каких-
либо ротамерных форм, не всегда приводит к отрицательным
при-экспоненциальным амплитудам. Мы делаем вывод о том,
что форма САЗ сама по себе недостаточна, чтобы опреде
лить среди других причину гетерогенности затухания трип-
тофановой флюоресценции в белках.
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УДК 577.32 + 577.322
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