Journal of Molecular Structure xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstruc

Binding phenomena and fluorescence quenching. II: Photophysics

of aromatic residues and dependence of fluorescence spectra on protein
conformation q
Patrik R. Callis ⇑
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, United States

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Survey of useful tryptophan models

for testing outside the protein
environment.
 Survey of natural quenchers and non-
quenchers of tryptophan found in
proteins.
 Introduction to principles behind why
excited Trp is easily quenched by
electron transfer.
 Introduction to why Trp is so
sensitive to electrostatic environment
whereas similar sensor molecules are
not.

a r t i c l e i n f o a b s t r a c t

Article history: The three amino acids with aromatic ring side chains—phenylalanine (Phe), tyrosine (Tyr), and especially
Received 15 April 2014 tryptophan (Trp) have played a long and productive role in helping unlock the secrets of protein behavior
Accepted 15 April 2014 by optical spectroscopy (absorption, fluorescence, circular dichroism, etc.) In principle, an appropriately
Available online xxxx
placed Trp will undergo fluorescence wavelength and/or intensity changes upon whatever functional
process a protein performs. Although perceived to be enigmatic and not well understood, Trp is arguably
Keywords: now better understood than many of the extrinsic probes currently in use. Basic principles of intrinsic
Protein
tryptophan fluorescence quenching and wavelength shifts in proteins are presented, with strong
Tryptophan
Fluorescence
emphasis on the importance of electrostatics. The condensed description of findings from recent
Quenching experiments and simulations of tryptophan fluorescence and intrinsic quenching in proteins is designed
Electron transfer to help authors in planning and interpreting experimental results of ligand binding studies.
Molecular dynamics simulations Ó 2014 Elsevier B.V. All rights reserved.

Introduction chains—phenylalanine (Phe), tyrosine (Tyr), and especially trypto-

Proteins are the working machines of life forms. As such, there
is intense interest in understanding the mechanistic details of pro-
tein function. The three amino acids with aromatic ring side
q
This article is part of a special issue titled ‘‘Fluorescence studies of biomolecular
association processes. Towards a detailed understanding of spectroscopic, thermo-
dynamic and structural aspects’’.
⇑ Tel.: +1 1 406 994 5414.
E-mail address: pcallis@montana.edu
phan (Trp) have played a long and productive role in helping
unlock the secrets of protein behavior by optical spectroscopy
(absorption, fluorescence, circular dichroism, etc.) [1–10]. This sec-
tion seeks to provide an in-depth, up-to-date resource about prop-
erties of these intrinsic aromatic residues that will familiarize
those planning to use fluorescence as a tool for proteins—especially
in the context of ligand binding.
Trp dominates the following presentation for the same reason
that Trp is the most widely exploited of the aromatic amino acids.
The larger p-system makes it the strongest and the most

http://dx.doi.org/10.1016/j.molstruc.2014.04.051
0022-2860/Ó 2014 Elsevier B.V. All rights reserved.

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2 P.R. Callis / Journal of Molecular Structure xxx (2014) xxx–xxx
red-shifted absorber and emitter, and both its emission wave-
length and quantum yield are quite sensitive to the local electro-
static environment (electric field direction) in proteins. Normally,
this means it is possible to exclusively excite Trp, thereby eliminat-
ing complexities due to FRET from Tyr and Phe. In principle, an
appropriately placed Trp will undergo fluorescence wavelength
and/or intensity changes upon whatever functional process a pro-
tein performs.
A primary theme of this paper is that understanding of fluores-
cence spectral shifts and quenching due to electron transfer
depends on having a basic (but not extensive) grasp of electrostatic
principles. To aid the reader, an Appendix is provided that gives
rapid access to useful numerical values in relation to spectral shifts
and energy gaps in terms of the extremely high electric fields and
potentials found in proteins.
Tryptophan
Trp has a reputation for being enigmatic and not well under-
stood. This was, indeed, once the case, but it is arguably now better
understood than many of the extrinsic probes currently in use
[1,10–57]. The just-cited references are a somewhat biased repre-
sentative selection from over 10,000 papers in the last 60 years
addressing tryptophan or indole fluorescence. Other papers cited
in this review are also significant.
The perceived complexity of Trp comes from the nature of the
pyrrole-benzene ring system which causes the two lowest excited
singlet states, 1Lb and 1La, to be accidently degenerate, unlike both
Phe and Tyr, which have widely separated 1Lb and 1La states [58].
For many years there was belief that either state might be the
emitting state, depending on the protein environment. An ad hoc
practice of assigning fluorescence with vibronic structure to 1Lb
has turned out to be merely the consequence that the large perma-
nent dipole change for the 1La transition (not to be confused with
transition dipole) leads to sufficiently large inhomogeneous broad-
ening in most protein environments that the natural vibronic
structure is obscured. Studies in cold vacuum have verified the pre-
dicted vibronic structure for the 1La transition [29], and several
studies have since shown that the more intensely absorbing, sol-
vent-sensitive 1La state is the emitting state in the vast majority
of proteins [27,29,48,59,60]. For only two mutant proteins showing
exceptionally blue shifted fluorescence has evidence of 1Lb emis-
sion been observed [48]. Even the extremely blue shifted fluores-
cence of Trp48 of azurin, whose fluorescence wavelength
maximum (kmax) is 306 nm, has been shown to emit entirely from
1
La [48,60].
Model reference molecules
Studies that depend on the quenching of a fluorophore in a
polymer will always be strengthened by collecting data under
identical conditions for model systems in the absence of the pro-
tein environment. We first identify and describe those most used
for Trp.
3-Methylindole (3MI)
3MI has only a methyl at the 3-position where Trp connects to
the protein backbone. This is a useful reference because the
absence of the backbone section eliminates intrinsic quenching
by electron transfer, while other spectroscopic properties are not
significantly affected. This is a better model than indole because
the 3-methyl substitution shifts the 1La state to lower energies,
allowing it to cross the 1Lb state and become the fluorescing state,
even in hydrocarbon environments, as noted above. The little-
cited, but unique and important study by Meech et al. [11] revealed
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that both the radiative and non-radiative rates for 3MI in solution
increase considerably as solvent polarity decreases. As a result, the
quantum yield remains relatively independent of polarity while
the lifetime decreases from 9 ns in water to 4 ns in cyclohexane,
with intermediate values in ether or alcohol. The radiative rate
increases at shorter wavelengths (non-polar solvents) for two rea-
sons: (1) all radiative rates are proportional to the cube of the fre-
quency of the light emitted, and (2) the transition dipole (the
square of which determines the radiative rate) increases with
decreasing polarity. The latter is because the excited state wave-
function has less charge transfer character in non-polar solvents.
These together explain the otherwise curious phenomenon that
sf for the buried, high quantum yield Trp48 of azurin is only 4 ns
whereas Trp in water-exposed positions may have an equally high
quantum yield with sf of 9 ns.
In contrast, the mechanism by which the internal non-radiative
rates (internal conversion and intersystem crossing) for 3MI
increase as the 1La absorption is blue shifted does not seem to be
known.
N-acetyltryptophanamide (NATA)
NATA in water is a good model for a solvent-exposed Trp in a
non-terminus protein site. This is because the two nearby amides
are chemically very similar to the two nearby backbone amides
that all protein Trps experience. The quantum yield is 0.14, about
half that of 3MI, virtually proving that electron transfer quenching
to backbone amides is a viable mechanism, as was recognized
quite early [46,61–63]. NATA in alcohols and ethers is a good
model for more buried Trps. In these less polar environments it
is found that electron transfer to the amides is suppressed because
the CT-1La energy gap fluctuations are smaller, not because the
energy gap is smaller [64]. An interesting and revealing study
has been performed by Alston et al. [65,66], demonstrating that
the local amino acid sequence affects Trp fluorescence in small
peptides and denatured proteins.
Tryptophan (free amino acid)
The free amino acid at any pH is not a good model for Trp in a
protein because there is no comparable bonding environment hav-
ing the charged ammonium and carboxylate groups tethered in
such close proximity to the indole ring.
Trp-containing dipeptides
These can be used to model cases in which Trp in a protein is in
the N- or C-terminal position [37,40].
5-Fluoro3MI, 5-fluoroNATA, and 5-fluorotryptophan
Whereas the previously listed models are useful for mimicking
Trp in various environments in proteins, their 5-fluorotryptophan
(5-FW) derivatives provide valuable information for why they gen-
erally do not mimic Trp in proteins. 5-FW has found considerable
use as a modified Trp that can be incorporated into proteins in
place of Trp. Broos and coworkers [67,68] found that when incor-
poration was near 100%, most Trps that showed multi-exponential
fluorescence decay curves became effectively mono-exponential
when replaced by 5FW. This striking behavior was subsequently
explained by the slightly higher (0.2 eV) ionization potential of
5FW compared to the model 3MI because the highest (energy)
occupied molecular orbital (HOMO) is stabilized by 0.2 eV [32].
That such a seemingly small decrease in electron donating power
can produce such a dramatic effect on the sensitively of the local
electrostatic environment strongly supports the discrete state
hypothesis underlying non-exponential fluorescence decay of Trp
in proteins. This modification has virtually no effect on the sensi-
tivity of wavelength to environment, so its use is valuable for
removing unwanted heterogeneity caused by sensitivity to
 P.R. Callis / Journal of Molecular Structure xxx (2014) xxx–xxx 3

electron-transfer based quenching while retaining nearly the same
wavelength sensitivity [43]. The quantum yield of 5-FW indicates
that 5-F substitution, for unknown reasons, does not inhibit
quenching by proton transfer [69].
Trps in proteins are usually ‘‘pre-quenched’’
Virtually all reported quantum yields of single Trps in proteins
fall in the range 0.35 down to 0.01 or less [1,6]. Recall that 0.35 is
essentially the quantum yield exhibited by 3MI in any solvent [11].
The interesting physical basis for this remarkable—and very use-
ful—variability will be discussed in Section 2.4. For now, we note
that the implications of this ‘‘pre-quenched’’ state of many of the
candidate Trps for sensing ligand binding must be kept in mind.
For an intrinsically quenched Trp with Uf = 0.15, for example,
ligand binding may actually increase the fluorescence by partially
or wholly removing the quenching mechanism, or may cause
pseudo-quenching, by enhancing the existing intrinsic quenching
mechanism, or by introducing a new one. These may come about,
for example, by local structure conformational change, electro-
static effects, changing the nature of water exposure, etc. This does
not compromise the use of Trp as a sensor, but merely means that
one cannot safely assume that the ligand is actually quenching,
even if it is capable of doing so.
One important example is the monitoring of Trp fluorescence
changes in myosin upon addition of ATP for fundamental studies
of muscle contraction. Because ATP itself does not quench Trp fluo-
rescence, an early study by Morales and coworkers concluded that
Trp fluorescence quantum yield could be affected by electrostatic
effects alone [70]. Similarly, there are numerous accounts of Trp
fluorescence changes upon binding one of the most important
ligands in biology, Ca2+, which is not a quencher [71].
For example, quenching of Trp214 fluorescence of human
serum albumin by peptides observed by Gonzalez-Beja and
coworkers [72] cannot be assigned with certainty to quenching
by the peptide binding, given the relative weakness of amides as
quenchers and the likelihood that structural changes accompany-
ing binding could alter the intrinsic quenching by the local protein
backbone near the Trp.
Quenchers of Trp models in water
Before tackling the more involved subject of intrinsic quenching
of Trp fluorescence in proteins, we consider the more straightfor-
ward information that exists for collisional quenching of 3MI in
aqueous solution by groups that are potential quenchers in
proteins.
There are two primary intrinsic quenching mechanisms at work
in the case of Trp: (1) quenching by electron transfer from the
excited indole ring to the nearby backbone amides or electron-
accepting side chains; (2) quenching by proton transfer to elec-
tron-rich atoms of the indole ring. The protons come from acidic
electron transfer-based quenchers of 3MI (and therefore of Trp,
in principle) commonly found in proteins. Note that the proton-
ation state is quite important. The second order collisional quench-
ing rate constant, kq, is also shown in Table 2 to indicate the
relative effectiveness of the quenchers [16]. Acrylamide, a common
collisional quencher used to sense whether Trp is solvent exposed
is included for reference [16].
Proton transfer-based quenching. Table 2 lists groups that are
found to quench indole and 3MI in water by a not so well under-
stood mechanism involving proton transfer to the 4, 7, or 2 posi-
tions of the excited indole ring [16,53]. Robb, Olivucci and
coworkers [74,75] have proposed a mechanism for this type of
quenching.
Proton-transfer based quenching is distinguished from electron
transfer-based quenching by its pronounced deuterium isotope
effect [16]. Intramolecular proton transfer from the ammonium
group of free Trp zwitterion at neutral pH reduces Uf to 0.14 rela-
tive to the value near 0.35 found for 3MI and Trp at pH 10 [14,76].
Intrinsic quenching in proteins
While considerable progress has been made towards under-
standing Trp lifetimes and quantum yields in proteins, the subject
remains complex for two reasons:
(a) There are ten groups in proteins that in principle can quench
Trp fluorescence.
(b) Potential quenchers of a Trp in a particular protein may or
may not actually quench; the extent of quenching depends
critically on the precise electrostatic environment for both
the Trp and the quencher—affecting whether electron trans-
fer is uphill or downhill in free energy.
The fluorescence quantum yield varies by 30-fold for single
Trps in different proteins. This has turned out to be because of
extreme variation among proteins regarding the ability of the local
electrostatic environment to assist or inhibit electron transfer to
the nearest backbone amide groups [31,77]. Such sensitivity to
quenching by electron transfer is the primary reason that Trp
fluorescence is widely used for monitoring so many protein
processes, including ligand binding. Only recently, however, was
the mechanism of this sensitivity rationalized [31,77]. Below,
we: (a) address why excited Trp is able to transfer electrons easily,
(b) why electron transfer quenches fluorescence, (c) what types of
environments favor or disfavor electron transfer, and (d) why Trp
is so sensitive to electrostatic environment whereas similar sensor
molecules are not.
Table 1
Electron transfer quenchers.
Quencher kq  107 M 1
s 1

side chains such as tyrosine, and the ammonium groups of lysine

Disulfide bonds >1000
and N-termini. Barkley and coworkers have made a careful study Acrylamide 720
of isotope effects on these quenchers that clearly distinguishes Histidine cation (pH 5.3) 240
between the two mechanisms by the higher deuterium isotope Cysteine anion (pH 10.4) 190
effect for proton transfer quenching [16]. Intrinsic quenching of Neutral cysteine (pH = 7.6) 140
Neutral acetic acid 43
Trp fluorescence by FRET, however, is typically not possible
Neutral glutamic acid (pH 4.5) 43a
because no other amino acid has an absorption spectrum that over- Neutral aspartic acid (pH 4.5) 43a
laps the Trp emission (except in rare cases of tyrosine anion). Neutral C-terminal carboxyl 43a
Electron transfer quenchers. In water, there is compelling exper- N-acetylasparagine 8.8b
imental evidence that the following groups can act as collisional N-acetylglutamine 6.5b
Protein backbone amides 0.1 to 500
quenchers of the pertinent model chromophore, 3MI, by an elec- Neutral histidine (pH > 6) 3.7
tron transfer mechanism [15,16,73]. Barkley and coworkers [16]
a
have carefully distinguished this mechanism from a separate Behavior is expected to be very similar to neutral acetic acid.
b
N-acetylation is to remove proton transfer quenching by N terminal NH+3.
mechanism due to proton transfer. Table 1 lists the important

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4 P.R. Callis / Journal of Molecular Structure xxx (2014) xxx–xxx

Table 2
Proton transfer quenchers.

Quencher kq  107 M 1
s 1

Neutral tyrosine side chain 98

N-terminal ammonium groups 26
Protonated lysine side chain 10

(a) Trp fluorescence is easily quenched because it can donate an

electron with relative ease. This is directly related to the fact
that Trp is one of the best reducing agents in biology. Both
facts stem from the remarkably low ionization energy of
the ground state (7.4 eV) compared to many aromatic sys-
tems, e.g., benzene (9.2 eV). This simply means an electron
occupying the HOMO of the Trp side chain lies high in
energy relative to that of most other molecules. Even in
the ground state, Trp acts as a reducing agent in some
well-known biological redox reactions. The lowest energy
excited state of Trp is 4 eV higher than the ground state,
meaning that an electron can be transferred with even more
ease—if the electrostatic environment is favorable.
(b) Why does electron transfer quench fluorescence? The
answer is that following electron transfer, neither the
indole ring radical cation nor the radical anion of the accep-
tor entity are able to fluoresce (they are ground state radi-
cals), and CT fluorescence that would reverse the electron
transfer is extremely weak. Furthermore, molecular dynam-
ics combined with quantum mechanical computation
(MD + QM) simulations suggest that solvent relaxation
about the CT state stabilizes the CT state to an energy well
below that of the 1La fluorescing state within 100 fs! There
is therefore little time for the electron to return, and the
electron remains trapped until a dark electron transfer back
to the indole ring returns the system to the ground state.
Fig. 1 shows MD + QM trajectories of computed excitation ener-
gies to the 1La and lowest CT states for two proteins bathed in
explicit water at room temperature. The energies are given in thou-
sands of cm 1, where 33000 cm 1 is 300 nm. Throughout the first
50 ps, the water and protein is equilibrated to the charge distribu-
tion of the 1La state. At the arbitrary time of 50 ps, the charges on
the Trp were changed to those of the CT state. The subsequent
rapid stabilization of the CT state in each case by 16,000 cm 1
(2 eV or 192 kJ/mol) is what appears to be the primary act of
quenching.
(c) What causes the variability? From early times the root basis
for most of the variability was believed to be electron trans-
fer from the excited indole ring to the closest backbone
amides [46,62,63]. A study of cyclic hexapeptides, designed
to contain no quencher candidates except the backbone
[78,79], incisively demonstrated that the backbone amides
are effective quenchers, and also showed strong variation
depending on sequence. The precise mechanism, however,
by which some Trps in proteins exhibited the full 0.35 value
for quantum yield while others had a quantum yield <0.01 in
the absence of obvious strong quenching side chains, was
unknown until 10 years ago. Even with X-ray structures, it
was difficult to understand why many Trps in proteins were
not quenched at all while others were strongly quenched,
given that all Trps are close to at least two backbone amides.
As noted above, the answer to this question lies in the ability
of the enormously strong electrostatic fields (potential dif-
ferences) typical of proteins to assist or inhibit the electron
transfer process to an amide. Electron transfer—as for all
processes in nature—requires that energy be conserved;
Fig. 1. Quantum mechanical excitation energies for the fluorescing state (1La (red)
and CT state (black), computed from molecular dynamics simulations for A. Staph.
nuclease; B. Dsba. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
the CT state and the fluorescing states must be in resonance,
i.e., must have the same energy at the instant of transfer.
This requires that positive charges are close to the acceptor,
and/or negative charges are near the donor.
This was first understood semi-quantitatively through
MD + QM simulations [31,77]. Fig. 1 compares representative
MD + QM results for Staphylococcus nuclease (STNase), whose sin-
gle Trp is very fluorescent, and Dsba, whose single Trp is weakly
fluorescent. The large CT-1La energy gap found for STNase is consis-
tent with a high quantum yield, because the CT state has little
probability to have the same energy as 1La. Likewise, the small
energy gap seen for Dsba offers much higher opportunity for elec-
tron transfer. Fig. 2 is a cartoon of why the energy gaps differ so
much between these two cases, and exemplifies the general under-
lying cause of the variability. Fig. 2A shows the specific case of
positive and negative groups, e.g., lysine and glutamate, stabilizing
electron transfer to a backbone amide. Such an arrangement will
lead to a weakly fluorescing Trp, in the absence of other nearby
charges. The opposite arrangement shown in Fig. 2B obviously
inhibits electron transfer to the amide, and leads to a highly fluo-
rescent Trp. This is an example of what could be called an internal
resonance Stark effect, similar to the external resonance Stark
effect described by Boxer and coworkers [80]. What is left out of
the cartoon is the considerable contribution from water molecules,
especially with regard to H-bonding to the amides.
(d) Why Trp is so sensitive to electrostatic environment was
alluded to in Section 2.1 on 5-fluorotryptophan [32]. This
has to do with the size of the CT-1La energy gap being such
as to give a quenching rate close to kr + knr in most proteins.
According to Eq. (1), the quantum yield would be near 0.15.
If the quenching rate is substantially increased or
decreased, the quantum yield will span the 0–0.3 range.
Fluorophores that have much larger or much smaller energy
gaps will have very low or very high quantum yields that
are less sensitive to small changes in the energy gap.

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 P.R. Callis / Journal of Molecular Structure xxx (2014) xxx–xxx 5

Fig. 2. How the location of environment charges (circled) determines the quenching by electron transfer to a backbone amide. The uncircled charges represent the dipole
created by electron transfer from the indole ring to an amide. In (A), charges stabilize the dipole of the CT state with electron transferred towards the positive and away from
the negative. The CT state is in resonance with the fluorescing (1La) state, electron transfer is fast, and fluorescence is weak. In (B), charges are arranged oppositely from case A,
greatly destabilizing the CT state. There can be little opportunity for resonance; quenching is therefore slight, and fluorescence is strong. Water and other polar groups are
equally important in contributing to the overall electrostatic influence on electron transfer in both cases. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

The general picture portrayed in Fig. 2 is valid, provided electro-
statics of H-bonding from water and other groups is included. It is
supported by considerable circumstantial computational evidence
that demonstrates the crucial importance of the local electric field
direction and magnitude in stabilizing the electron occupation of
the empty p orbital localized mostly on the amide carbonyl car-
bon [17,31–34,41,54,77,81–85]. This striking variability is not
due to differences in the local polarity because the quantum yield
is nearly independent of solvent polarity for indole chromophores
[11]. Electron capture into the same p orbital has been strongly
implicated for fragmentation mechanisms in mass spectroscopy
of peptides [86]. Recently, Parson, Anderson and co-workers were
able to predict fluorescence quantum yields for Trp in several
folded hairpin peptides using a similar, but independent MD + QM
method [54].
An example of how electrostatic effects can cause unexpected
results comes from an extensive study of the conserved, weakly
fluorescing Trps 68 and 156 of human gamma D and gamma S
crystallin mutants, in which Chen et al. [33] replaced Cys32 whose
S is only 4 Å distant from Trp 68 ring atoms. No increase in quan-
tum yield was found, however, apparently because of negative
groups near the cysteine, which suppress its electron accepting
power. In other cases, there is evidence of moderate quenching
by Cys [67,87].
In another example, histidine cation was documented as a
quencher in proteins, typically by showing a decrease in Uf upon
lowering pH [88,89]. For the Q105H mutant of T4 lysozyme, how-
ever, Van Gilst and Hudson [52] found that lowering the pH
increased Uf of the nearby Trp138. We have argued that this seem-
ingly anomalous behavior is caused by the electrostatic effect of
the His cation positive charge near the indole ring, which reduces
the already substantial quenching by the backbone amide, but not
enough to send the electron to the His [31]. This behavior illus-
trates the difficulties of predicting Trp fluorescence quenching
pathways in proteins.
Proton transfer quenching in proteins. There is much less evi-
dence for proton transfer quenching compared to that from elec-
tron transfer in proteins. There is often association of Trp with
Lys or Arg in what are called cation-p complexes, but Trps in such
complexes are almost always highly fluorescent. Apparently, in
such complexes, the ammonium group of the Lys is either con-
formationally hindered from donating a proton, or a more aqueous
environment is required. The high Uf values in these complexes are
easily understood in terms of electrostatics, as indicated in Figs. 1
and 2. Close proximity of neutral tyrosines to Trps is common in
proteins, but does not seem to correlate with quenching.
Quenching by some ligands in proteins
Trp fluorescence quenching by ligands have provided useful
insights. A few are mentioned here, including quenching by heme,
Cu2+, Eu3+, and Yb3+.
Tryptophan fluorescence is highly quenched in heme-contain-
ing proteins. Hochstrasser and Negus found Trp fluorescence life-
times on the order of 10 s of ps for several myoglobins, in which
the separation between Trp and heme is 20 Å [90]. Their analysis
for Sperm Whale and Tuna myoglobins assuming quenching was
solely by FRET gave results in agreement with experiment.
Trp 48 of azurin has one of the most hydrophobic environments
known, leading to extremely blue-shifted fluorescence and maxi-
mally high fluorescence quantum yield in the absence of Cu II. In
its active form, a Cu II ion is bound in a site 10 Å from the closest
ring atom of Trp48, and quenches the fluorescence by 20-fold. A
thorough study of azurins from different species by Petrich et al.
tentatively assigned the quenching mechanism as electron transfer
from the excited Trp48, reducing the Cu II to Cu I.
Horrocks et al. showed definitively that electron transfer occurs
over 10 Å in cod parvalbumin when the Ca2+ binding sites are occu-
pied by Eu3+ or Yb3+ [91,92]. In the lanthanide series, Eu3+ and Yb3+
have by far the most positive reduction potentials, and are the only
lanthanides that quench Trp fluorescence when bound to cod par-
valbumin. Electron transfer was shown as the only viable mecha-
nism for Yb3+. FRET is ruled out because Yb3+ has no absorption
bands that overlap Trp emission.
Nonquenchers in proteins
Chen and Barkley found no detectable quenching of 3-methyl-
indole fluorescence in water at neutral pH by N-acetyl derivatives
of glycine, alanine, valine, leucine, phenylalanine, proline,
hydroxyproline, serine, threonine, methionine, and arginine. The
lower limit for detection was kq < 1  107 M 1 s 1. Early quenching
studies reported that most of these as free amino acid zwitterions
quenched tryptophan fluorescence at pH 5–6 [93,94]. The use of N-
acetyl derivatives, however, subsequently revealed that in many
cases the unacetylated amino acids quenched by proton transfer
from the N-terminal ammonium group [16].
For emphasis and clarity, additional comments are made con-
cerning glutamate, aspartate, and phenylalanine, which are some-
times stated to be quenchers, but for which there is little or no
evidence that these can act as quenchers in solution or in proteins.
Glutamate and aspartate, being negatively charged, almost cer-
tainly cannot quench by accepting an electron. All charged groups,

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6 P.R. Callis / Journal of Molecular Structure xxx (2014) xxx–xxx
however, may appear to be quenchers in the protein environment
because of electrostatic effects. For example, a glutamate near the
indole ring of Trp will greatly assist electron transfer to a backbone
amide. Confusion may also arise because of the variability of pKa
values of glutamic and aspartic acids in proteins, which have been
observed as high as 7.5, i.e., 3 units above the normal values. Thus,
when somewhat buried, glutamate and aspartate may quench at
pH7, but only because they are protonated in that environment.
A conserved phenylalanine (Phe8) juxtaposed to Trp48 in the
large class of homeo domains was implicated as the reason for
the very low quantum yield of this Trp in non-polar environment
[95–97]. This appears to be a case of correlation instead of cause.
Nominally, the HOMO of benzene is too low in energy and the low-
est unoccupied MO (LUMO) is too high to facilitate quenching of
Trp. The picture is clouded by the fact that benzene quenches
Trp fluorescence in concentrated solutions. Subramanian et al.,
however, found that the Trp quantum yield remained very low in
the F8A mutant of Bicoid-homeo domain, somewhat dispelling
the notion that Phe is the quencher. There is MD + QM evidence
[98] indicating that the ubiquitous strong quenching of the con-
served Trp 48 in homeo domains is more likely due to the back-
bone amide, facilitated by a large helix dipole, whose field
strongly directs electrons from the Trp ring to the amide. Similarly,
the low quantum yield of conserved Trps 68 and 156 of cD and cS
crystallins from many species all have fluorescence quantum yields
near 0.01, yet extensive mutation of nearby plausible quenchers
fails to increase the quantum yield [33,34]. Similar to the homeo
domains, the conformation and surroundings result in efficient
quenching by the nearby backbone amide, helped considerably
by firmly trapped water molecules that stabilize the CT state.
Tryptophan and tyrosine are quenchers of dyes and flavins
Because of the high-lying HOMO energy, ground state Trp can
also act as a quencher of many fluorophores, e.g., flavins and dyes
[99–101]. When the HOMO of the dye lies substantially below that
of Trp, the vacancy in the HOMO of the excited dye may be filled by
an electron from the ground state Trp HOMO. (To a somewhat les-
ser extent, tyrosine quenches dyes and flavins by the same mecha-
nism.) The final state of the Trp ring immediately following
electron transfer is the same as when excited Trp is quenched by
an electron acceptor (the indole ring is a ground state radical cat-
ion and the dye is a ground state radical anion). Solvation behavior
following electron transfer is much the same as when excited Trp
donates an electron [82]. Because the HOMO of 5FW is lower than
that of Trp, 5FW is not expected to quench dyes as efficiently as
Trp.
Wavelength variation and Trp–Trp FRET
Although focus has been on quenching in the above sections,
there are cases for which binding induces quantifiable changes in
fluorescence or absorption spectra with little or no quenching. This
becomes especially useful when isosbestic points are associated
with binding [102–104].
As with electron transfer-induced quenching, electrostatics
plays a critical role in determining fluorescence emission shape
and wavelength maximum (kmax) [30,84,105]. Numerous quantum
mechanical studies on indoles have been summarized as unani-
mously agreeing that the transition from the ground to the 1La
state moves substantial electron density from the pyrrole ring to
the benzene ring [26]. Therefore, positive charges near the benzene
ring will contribute a shift of the absorption and fluorescence to
longer wavelengths (red shift) while positive charges near the pyr-
role ring will contribute a blue shift. Just the opposite will hold for
negative charges.
Please cite this article in press as: P.R. Callis, J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2014.04.051
A serious caveat, however, is that the strong effect of nearby
protein charges will be considerably offset by dielectric compensa-
tion from large numbers of water molecules out to 15 Å, which
may be partially oriented by charges near the chomophore. Only
when these very substantial contributions are included will mod-
eling predictions be accurate [30]. A spectacular example is the
case of Trp140 in STNase, which is flanked by two lysine ammo-
nium groups over the benzene ring. The nearby positive charges
nominally would contribute a 90 nm Coulombic red shift, but
mutations of these lysines to neutral sidechains is experimentally
found to result in only tiny (0.5 nm) shifts of the fluorescence
kmax [87]. This effect is well reproduced by MD + QM modeling
wherein polarization of distant water contributes compensating
blue shifts that in each case leave the resultant shift nearly
unchanged [35].
Water–protein relaxation. Shifts of absorption spectra are much
smaller than those of fluorescence because the ground state per-
manent dipole is much smaller than the 1La permanent dipole,
although both point in roughly the same direction (with the pyr-
role ring being more positive than the benzene ring). Immediately
upon excitation the system is in a non-equilibrium state, and the
surroundings begin to respond in a way to return to electrostatic
equilibrium. The largest component of this relaxation typically is
from water, because of its fast rotational and translational mobility
[105]. For solvent exposed Trps in proteins, the longest kmax is
352 nm, considerable shorter than that of 3MI in water
(374 nm) and NATA (362 nm) [14]. This is apparently because in
proteins, one side of the indole ring is shielded from solvent.
In recent years, the rate of this relaxation has been thoroughly
studied with ultrafast time resolved fluorescence upconversion
methods [38,39,41,49,50,106,107]. The timescale of the relaxation
is almost always observed to be biphasic, with the bulk of relaxa-
tion being completed within 2 ps, and a smaller component
relaxing over 10 ps ? >1 ns [43]. There is ongoing discussion con-
cerning whether protein, water, or both are responsible for the
slower times [35,107,108].
Trp–Trp FRET. When multiple Trps are present in a protein, there
is a high probability of Trp–Trp FRET, given that differences in elec-
trostatic environment will favor net unidirectional FRET to Trps
with more red-shifted fluorescence. An example is bacteriophage
T4 lysozyme, which has three Trps approximately at the corners
of an equilateral triangle with 15 Å sides. Observing phosphores-
cence and optically detected magnetic resonance at 4.2 K of the
wild type and all single and double Trp ? Tyr mutants, Ghosh
et al. [109] demonstrated that most of the excitation to W126 is
transferred to W158.
Tyrosine and phenylalanine
Fluorescence from tyrosine (Tyr) and phenylalanine (Phe) is
also a valuable resource for investigating protein function, but
has seen far less use than that of Trp. This is because these absorb
at shorter wavelengths, have weaker absorptivity, have less wave-
length sensitivity to environment, and transfer much of their exci-
tation to Trp if present nearby. In addition, both are far more
abundant than Trp in most proteins.
Some useful miscellaneous facts are that the low quantum
yields of Tyr and Phe in proteins is almost certainly due to quench-
ing by electron transfer to the local backbone amides, just as for
Trp [110]. An important fact associated with Tyr is that in certain
environments, or at high pH, tyrosinate may be present. This spe-
cies absorbs and emits near where Trp does, and can have a broad
red shifted emission similar to Trp.
Nevertheless, for proteins with no Trp and a limited number of
Phe and/or Tyr, circumstances exist for which Tyr and Phe fluores-
 P.R. Callis / Journal of Molecular Structure xxx (2014) xxx–xxx
cence can be exploited to obtain useful information. Some exam-
ples have been reviewed recently [2]. Although Phe fluorescence
is very difficult to detect in the presence of Tyr and Trp, it becomes
quite useful for those proteins not containing Tyr and Trp [2]. One
important protein meeting this criterion is calmodulin, for which
Shea and co-workers [111] have made useful studies of Ca2+ bind-
ing. These studies found a >50% decrease in the combined fluores-
cence of the 5 Phe residues upon binding one Ca2+. Given that Ca2+
is not a quencher, this is a case in which the structural change and/
or electrostatic effect has enhanced the normal quenching by elec-
tron transfer to amides.
Conclusions
When using tryptophan (Trp) as the fluorophore in a protein,
one should keep in mind the dozen or so potential intrinsic
quenchers, and the consequences that binding of an external
quenching ligand may have on any pre-existing intrinsic quench-
ing. Binding of a ligand near a Trp may enhance or reduce intrinsic
quenching by a direct electrostatic effect or indirectly by changing
the local structure, water exposure, etc. This is mentioned primar-
ily to help investigators avoid unwarranted conclusions regarding
the mechanism for the fluorescence change. For example, Ca2+
and ATP binding cause useful Trp fluorescence changes, but neither
can quench Trp fluorescence. The binding constant determined by
the fluorescence change should not depend on the exact mecha-
nism by which the fluorescence changes, however.
The effect of temperature upon quenching will always contain a
large component from the temperature dependence of fluores-
cence from the indole ring itself. The quantum yield of fluorescence
from 3-methylindole in water drops rapidly with increasing tem-
perature by a mechanism that is still not understood. Care must
be taken, therefore, when interpreting the meaning of the temper-
ature dependence of quenching by ligand binding.
Acknowledgements
The author is extremely indebted to Drs. Mickey Schurr, Bruce
Hudson, Dan Harris, Andy Albrecht, Tom Scott, Mary Barkley, Bob
Woody, Lennie Brand, Lee Spangler, Jaap Broos, Sandy Ross, Jay
Knutson, Franck Prendergast, and Dongping Zhong for helpful
interactions.
Notes on electrostatics
Potential has the units of volts, i.e., joules/coulomb. An electron
has lower energy in a more positive potential (closer to positive
charge or farther from negative charge). From Coulomb’s Law,
one can show that at a point 1 Å distant from a proton, the poten-
tial is +14.40 V. Therefore the energy of an electron 1 Å from a pro-
ton is 14.40 eV or 332 kcal/mol or 1389 kJ/mole. The electric
fields in proteins are on the order of a few times 107 V/cm, i.e., a
few tenths of V/Å [112,113]. The permanent dipole change during
excitation (not to be confused with transition dipole) to the 1La state
of Trp is 5–10 Debyes, meaning that a field of 107 V/cm can cause a
10 nm shift in the fluorescence.
Electron transfer-induced quenching
For electron transfer, if the amide C atom of Trp is more positive
than the indole ring by 1 V, the energy to create the CT state will be
1 eV = 8000 cm 1 = 23 kcal/mol = 96.5 kJ/mol less than if there
were no potential difference.
Please cite this article in press as: P.R. Callis, J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2014.04.051
7
Wavelength shift
For back-of-envelope calculations, one may use the shift of elec-
tron density for excitation to 1La as 0.25 electron from the pyrrole
to benzene ring. Therefore if the average potential of the benzene
ring is 1 V more positive than the pyrrole ring, the energy differ-
ence will be about 0.25 eV = 2000 cm 1, which is a 20 nm
red shift for a wave length of 320 nm. If the average potential
of the benzene ring is 1 V more negative than the pyrrole ring,
the energy difference will be about +0.25 eV = +2000 cm 1, which
is a 20 nm blue shift at 320 nm.
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