STUDY OF TIME-DEPENDENT

FLUORESCENCE STOKES
SHIFT OF MONELLIN

A Proposal

Submitted to the Central Department of Physics,

Tribhuvan University, Kirtipur in the Partial Fulfillment for the
Requirement of Master’s Degree of Science in Physics

By
........................
Bibek Tiwari
Roll No: 19
November, 2017
Contents

1 Introduction 1

1.1 Fluorescence and the Time-Dependent Stokes Shift . . . . . . . . . . . . . . . . . 2

1.1.1 Insight into the molecular probe . . . . . . . . . . . . . . . . . . . . . . . 4

2 Literature Review 4

3 Objectives 6

4 Methodology 6

4.1 Molecular Dynamics Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.1.1 Strategy of Force Field in the simulation . . . . . . . . . . . . . . . . . . . 6

4.1.2 Gradual development of force fields . . . . . . . . . . . . . . . . . . . . . 7

4.2 Time Dependent Fluorescence Stoke’s Shift (TDFSS) . . . . . . . . . . . . . . . . 8

5 Work Plan 8
1 Introduction
With the advent of life on earth roughly about four billion years ago, simple primordial organ-
isms originated with a potential to extract energy from chemical compounds and from sunlight can
be considered to be the cause of the currently complex molecular aggregation vital to life called
Biomolecules. Carbohydrates, lipids, nucleic acids, and proteins are the chief categories. The
study and analysis of structures of protein, thousands of different kinds of which are found even in
a single cell have infact an immense role in structure of body and also in extrapolating the genetic
information [1]. Similarly, the substantial job of sugars and particularly the sweetness character-
istic has to be seriously acknowledged, the intake of which are the part of our everyday life. The
chief sources of which comprises of Sucrose, Fructose, Honey and their derivatives. The major
pitfalls in them are the associated various nutritional and medical problems that can be linked to
great extent as a direct outcome of consumption of highly sweet, caloric and cariogenic sugars like
Sucrose [2]. Moreover the intake of artificial low caloric sugars also have numerous side effects
[3, 4]. Monellin firstly reported as a carbohydrate and later confirmed as a protein can be the sub-
stitute for avoiding the medical issues caused by sugars and also acts as a natural low calorie and
non-carbohydrate sweetener.

Amino acids are the common structural foundation on

which the protein molecules are designed by the nature:
the common structure containing a carboxylic acid group
(-COOH), an amino group (-NH2 ) attached to the first
(alpha-) Carbon (C), a Hydrogen atom (H) and a side
chain (R) that characterizes among them [1]. The poly-
merization of amino acids feasible due to some sort of
condensation reaction form chains whereby the peptide
bond, a linkage between CO-NH results yielding dipep-
tides, tripeptides, oligopeptides and polypeptides. In gen-
eral, at least 40 residues are involved in the protein for-
mation and the polypeptides consisting residues number
smaller than these are usually termed as peptides [6].
Twenty amino acids are involved in the mammalian pro-
teins and their single/triple letter(s) acronym are used to
represent the structures. The protein structure conforma-
tions are usually categorized as primary, secondary, ter-
tiary and quaternary in a hierarchical manner as shown Figure 1: Levels of protein
in Figure.1. Primary structures deals with the description structures [5].
of all covalent bonds (mainly peptide bonds and disul-
phide bonds) linking amino acid residues in a polypeptide chains. The arrangement of amino acid
residues for structural patterns is described by secondary structures: most commonly observed
structures being α-Helix and β-Sheet. The three dimensional folding of a polypeptide is described
as tertiary structure while such 3D spatial arrangement of protein with multiple polypeptides sub-
units is referred to as quaternary structure.

1
The protein Monellin (Molecular Weight 10.7 KDa ) acquired its name in 1972 following the
isolation and characterization in Monell Chemical Senses Center in Philadelphia, U.S.A. although
the discovery in the fruit of the west African Shrub known as serendipity berry (Dioscoreophyllum
cumminsii) of the family Menispermaceae was made already in the 1969 [7].
The secondary structure of Monellin consist of five
beta-strands that form an antiparallel β-sheet and a
17-residue α-helix bonded non-covalently [7]. Fig-
ure.2 shows some of the features so discussed: the
alpha-helix and beta-sheet are clearly seen.
Despite the fact that it is many thousands times (on
molar basis) sweeter than the commonly used sug-
ars along with the utility for sugar-related medically
challenged individual, the protein in its natural form
suffers from denaturation over 500 C and is highly
sensitive to pH and further in natural form the per-
ception of the sweet taste is somewhat delayed and
also suffers a persistent lingering sweet taste and a
slight after-taste [8, 9]. Nevertheless, the chemically
engineered form with long stability over temperature
& pH and appropriate mixing with other compounds
to reduce such lingering makes it always a good sub-
stitute for our use [8]. Figure 2: Snapshot of Monellin’s structure
The molecular composition of Monellin’s protein at 2.3 A0 resolution [pdb entry 4MON].
in its two chains following the single letter code
acronym for amino acids is:

Monellin chain A (44 AA):

REIKGYEYQL YVYASDKLFR ADISEDYKTR GRKLLRFNGP VPPP [10]
Monellin chain B (50 AA):
GEWEIIDIGP FTQNLGKFAV DEENKIGQYG RLTFNKVIRP CMKKTIYEEN [10]
Our research work studies the Fluorescence phenomenon of Monellin. We present a brief intro-
duction to the phenomenon of Fluorescence.

1.1 Fluorescence and the Time-Dependent Stokes Shift

The term “Fluorescence” coined by Stokes relates to an emission from excited singlet states while
that of Phosphorescence considers the identical phenomenon from triplet excited states [11]. The
former consists of electron in excited orbital paired (by opposite spin) to the electron in the ground
state orbital while the latter possesses the same spin orientation in both the states.

2
 (b) A schematic illustration of the time-dependent
Stokes shift (exaggerated for clarity). As time pro-
gresses, the fluorescence emission wavelength shifts
to the red (lower frequency) [12].
(a) One form of Jablonski diagram [11].

Figure 3: Jablonski diagram and the the scheme of TDSS.

Jablonski diagram as shown in Figure.3a usually appears to illustrate the process of absorption and
emission of light encountered by fluorophore, a fluorescent chemical compound that can re-emit
light upon light excitation [11]. The fluorescence emission occurs after the excited fluorophore in
the excited state, S1 quickly (10−12 sec) relaxes to its lowest vibrational state by the process called
internal conversion and the likewise phenomenon of emission from the state T1 , a triplet state de-
veloped as a consequence of intersystem crossing causes the phosphorescence [11].
One can easily infer from the Jablonski diagram that the fluorescence emission is relatively at the
lower energy in comparison to the excitation energy. This phenomenon of energy losses, first ob-
served by George G. Stokes is largely attributed to the rapid decay to the lowest vibrational level
of S1 and further enhanced when the de-excitation in higher vibrational level of S0 suffers the
energy loss by thermalization of such excess vibrational energy. The so called Stokes Shift in this
context is the difference (in wavelength or frequency units) between positions of the band maxima
of the absorption and emission spectra of the same electronic transition. Stokes shift of appropriate
molecular probe (discussed in brief in section.1.1.1) featured with significant changes in the dipole
moment in an excited state is studied and the time dependent shift of fluorescence emission max-
imum (Stoke’s Shift) is plotted considering at ν(t=0) immediately after the excitation continuing
upto say ν(t=∞) to the regions of longer wavelengths back to an equilibrium configuration before
an excitation. The peak center as a function of time is used to estimate Spectral Shift Correlation
function, S(t) given as Eq.1.

ν(t) − ν(∞) Esolv (t) − Esolv (∞)

S(t) = = (1)
ν(0) − ν(∞) Esolv (0) − Esolv (∞)

where Esolv (t)is the time-dependent energy response as measured by the probe at time ‘t’.

3
1.1.1 Insight into the molecular probe

(a) Snapshot of Tryptophan residue (cyan in color) in

Monellin protein.

(b) Absorption and Emission Spectra in pH 7 aqueous

solution [11].

Figure 4: Visualization of Tryptophan residue in monellin and its emission & absorption spectras.

Study of Protein Fluorescence, (Monellin for our current study) can be achieved via analysis of
suitable fluorescent agent (optical probe) since in general biomolecular compounds like lipids,
membranes, saccharides, etc are essentially non-fluorescent. In protein however, three aromatic
amino-acids phenylalanine, tyrosine and tryptophan being fluorescent makes us using them as a
suitable probe agents and thus finds great utility in spectroscopic studies. The underlying reason
is that our probe should display significantly higher dipole moment in the excited state over over
the ground state, Indole are reported with such features and its derivatives Tryptophan thus comes
in this field [11]. The graph shown in Figure.4b clearly shows the spectra of Phenylalanine, Tyro-
sine and Tryptophan. We thus realize the utility of Tryptophan for the selective excitation in the
wavelength range from 295-305 nm which is also the commonly used region so as to avoid the
controversy that may arise due to the overlap of the other two spectral data [11]. Using VMD we
have in the Figure.4a the clear visualization of Tryptophan residue in the monellin’s structure.

2 Literature Review
The solvation dynamics for Room Temperature Ionic Liquids has been studied with the aid of
Time-Dependent Fluorescence Stokes Shift (TDFSS) indicating relatively small stokes shift ex-
tending well into the regime of nanosecond scale[12, 13]. As a matter of fact, the rate of chemical

4
reaction in liquid is influenced by time scale of the response of solvent molecules to electronic re-
arrangement of solute molecules [14]. The femtosecond (fs) solvation dynamics of coumarin salt
in water revealed experimentally in conjugation with simulation that the librational (vibrational)
motions of solvent molecules cause the initial ultrafast response followed by the slow component
as a result of diffusive motions [12]. This remarkable prediction possible via the calculation of
stokes shift further demonstrated that the aqueous solvation dynamics seems to be dominated for
the timescale factor faster than 50 fs [14]. The molecular dynamics simulation study along with
the earlier experiment for the excited state relaxation dynamics of Lys-Trp-Lys reflects several
time scales ranging from femtoseconds to tens of picoseconds for inertial dynamics, for charges of
peptide and water re-equilibration in excited state and for shifting of relative population of three
isomers in the excited state [15]. Several experimental observations related to TDSS has been made
in the fluorescence of solvatochromic dyes in polar solvents and also in the fluorescence of solva-
tochromic flurophores involved in biological macromolecules such as protein and DNA [16]. Sol-
vent Dielectric Dispersion data can be used in estimating the time dependent fluorescence stokes
shift [13]. Using CHARMM (Chemistry at Harvard Macromolecular Mechanics) force field with
certain parameterization the molecular dynamic simulation study was done to examine the molec-
ular origin of the Fluorescence Stoke’s Shift from a tryptophon residue in a protein [17]. However
no general consensus on the origins of TDSS in biological macromolecules has been reached.

(a) UV abs.spectrum of monellin. (b) Fluorescence emission spectra of monellin.

Figure 5: Absorption 5a and emission spectra 5b of monellin [18].

Various research activities have been done in case of the Monellin protein on the theoretical
and experimental basis. Figure.5a shows the Ultraviolet absorption spectrum measured with a Cary
14 recording spectrophotometer at room temp (220 C) giving the maximum absorption at a wave-
length of 277 nm. The fluorescence emission as shown in Figure.5b is obtained by the excitation at
a wavelength of 277 nm measured with a Perkin-Elmer model MPF-2A fluorescence spectropho-
tometer at 200 C at a protein concentration at 0.1 mg per ml in 0.1N NaOH. An engineered variety
of monellin’s single chain has been successfully studied with the aid of Nuclear Magnetic Reso-
nance (NMR) [19]. The hydration dynamics of Monellin was studied by a group of researchers
using parameterized CHARMM force fields embedded in a 28 A0 radius sphere of TIP3P water
model propagated using Langevin integrator. The underlying time step integration was done using

5
Leap-Frog integrator while the bond restriction was done with SHAKE algorithm [20]. We find
that the simulation study of the Monellin with the currently optimized several parameters has not
been done so far. Newly discovered physics are encoded in it which can help us to gain some
deeper insight into the study of the Monellin structure i.e. we study the time-dependent fluores-
cence stokes shift of Monellin (at resolution of 2.3 A0 ) obtained from Protein Data Bank (pdb
entry 4MON) using the Molecular Dynamics simulation. We attempt to obtain the experimentally
valid results using the simulation and study other various properties which is difficult to perform
via direct real-time lab experiment. We use the optimized force fields, bond restriction algorithms
and water model currently recognized to be as well suited for the simulation works.

3 Objectives
• To gain insight into the dynamics of photoexcitation of biomolecules.

• To understand the role of timescales in various levels of solvation dynamics.

• To develop ideas regarding the origin of TDFSS in macromolecules.

4 Methodology
Our research work is based on the computer simulation. The computer simulation has been widely
used as parallel to the real time-experiment for scientific research. We utilize the tools of Molec-
ular Dynamics simulation to conduct our research activities. Some basic aspects underlying this
approach is discussed below:

4.1 Molecular Dynamics Simulation

Analysis of mesoscopic system of biological significance is vital in understanding the life pro-
cesses. The phenomenon of jiggling and wiggling of atoms, molecules, etc governs the mechanism
of living systems and thus applying the atomic/molecular approaches helps in reducing the com-
plex biological functionalities in the regime of physics and chemistry [21, 22]. The description of
most of the properties can be made using the tools of classical mechanics. The so called Molecular
dynamics simulation utilizes the classical mechanics even for the nuclear motion for analyzing the
classical many body system to predict and estimate the various transport and equilibrium properties
of interest. It generates a phase space trajectory applying the notion of Newtonian Mechanics. In
reality though the method in itself is not an entire approach to develop the theoretical model, they
only facilitates as the bridge between the experiment and theoretical model and play a fundamental
role in understanding the essential components of the living system from the microscopic point of
view disregarding the quantum effects.

4.1.1 Strategy of Force Field in the simulation

Force Field in Molecular Dynamics Simulation refer to the mathematical expressions that relate
the dependence of the energy of a system as a function of the coordinate of its particles. The

6
interactions between particles (typically atoms) can be described by the set of so called potential
functions and the parameters involved in it. Results compiled from ab initio or semi-empirical
quantum mechanical calculation and the experimental data related to Neutron, X-ray, electron
diffraction, etc are involved in the parameterization of the force fields [23].

Figure 6: Continuum model: Showing interaction function in modern force fields [24].

The potential functions subdivides as bonded and non-bonded interactions. Covalent bond-stretching,
angle-bending, torsion potentials when rotating around bonds, and out of plane “improper torsion”
potentials are major sub categories under the bonded interaction while the non-bonded interaction
that are based on neighbor list consists Lennard-Jones repulsion and dispersion along with the
Coulomb electrostatics. The typical expression for force fields with symbols carrying usual mean-
ings resembles as Eq.2 [23]. The major drawback of such harmonic function approximation in the
force field it that the bond cannot be broken thereby no chemical processes can be studied.

X 1 X 1 X Vn
U = Kb (r − r0 )2 + Ka (θ − θ0 )2 + [1 + cos(nφ − δ)] +
bonds
2 angles
2 torsions
2
"   6 #
X qi .qj 12
X X σij σij
Vimp + + 4ij − (2)
improper elec
rij LenardJones
rij rij

4.1.2 Gradual development of force fields

The spectroscopic or crystallographic structural data available contributed in the development of

the first generation of classical bio-molecular fields such as AMBER (Assisted Model Building
with Energy Refinement), CHARMM, OPLS-AA (Optimized Potentials for Liquid Simulations)
and GROMOS (GROningen MOlecular Simulation). The simulation of liquids possible with in-
creasing computing facility so that the various condensed phase thermodynamic data such as den-
sity, energies and free energy when included in the parameterization led to the second generation
of force fields. GROMOS 45A4, GROMOS 53A6 thus developed included the parameterization

7
necessary for the case of aliphatic chains, the polar amino acid side chains and peptides. However
an experimental discrepancy over the stability of α-chains forced to implement new consideration
in torsional parameters [25]. This re-parameterization resulted into GROMOS 54A7, the reparam-
eterization of which was largely based on the experimental evidence of N-O bonded interaction
between peptides. Furthermore, this force field works as a valid representation regarding mixed
α-β peptides molecules. Hence, for our research work of Monellin GROMOS 54A7 is good ap-
proximation.
Further for an effective simulation works various algorithms have been developed to facilitate the
integration of equations of motion, constrained bond dynamics, updating water models, etc. The
finite system under consideration often encounters a different force scenario at the boundary edges
and the implementation of periodic boundary conditions helps in resolving such issues [26].

4.2 Time Dependent Fluorescence Stoke’s Shift (TDFSS)

The time-dependent fluorescence stokes shift can be computed with the help of computer simula-
tion considered over the given ensemble in normalized form as,

< ∆Ef l (t) − ∆Ef l (∞) >

S(t) = (3)
< ∆Ef l (0) − ∆Ef l (∞) >

where

∆Ef l (t) = Eex (t) − Egr (t) (4)

Eex (t) and Egr (t) represents in the excited and the ground states respectively. ∆Ef l (∞) is taken
from an average of ∆Ef l (t) over prescribed time for simulation.

5 Work Plan
Time Duration (in months)
Work
1-2 3-4 5-6 7-8 9-10 10-11
Literature
Review
Software
Familiarization
Data enumeration &
calculation
Data Analysis &
Paper publishing
Thesis writing &
Documentation

8
References
[1] D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, W. H. Freeman, New
York (2008).

[2] http://www.who.int/oral health/disease burden/global/en/, (Accessed on 11/07/2017).

[3] S. Cohen, Health News, 7, 10 (2001).

[4] R. B. Kanarek, Nutr. Rev., 52, 173 (1994).

[5] http://www.particlesciences.com/news/technical-briefs/2009/protein-structure.html, (Ac-

cessed on 11/07/2017).

[6] D. Voet and J. G. Voet, Fundamentals of Biochemistry, Wiley, N J (2010).

[7] G. Bujacz, M. Miller, R. Harrison, N. Thanki, G. L. Gilliland, C. M. Ogata, S. H. Kim, and

A. Wlodawer, Acta Crystallogr., 53, 713 (1997).

[8] Burge et al.,Sweetening Compositions Containing Protein Sweeteners (1976).

[9] N. C. Kim and A. D. Kinghorn, Arch. Pharm. Res., 25, 725 (2002).

[10] https://www.rcsb.org/pdb/explore/explore.do?structureId=4MON, (Accessed on

11/07/2017).

[11] J. R. Lokowicz, Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York
(2006).

[12] https://web.stanford.edu/group/fayer/research stokes.html, (Accessed on 11/07/2017).

[13] C.-P. Hsu, X. Song, and R. A. Marcus, J. Phys. Chem. B, 101, 2546 (1997).

[14] R. Jimenez, G. R. Fleming, P. V. Kumar, and M. Maroncelli, Nature, 369, 471 (1994).

[15] A. A. Hassanali, T. Li, D. Zhong, and S. J. Singer, J. Phys. Chem. B, 110, 10497 (2006).

[16] D. Toptygin, in Reviews in Fluorescence 2015, edited by C. D Geddes, Time-Dependent

Spectral Shifts in Tryptophan Fluorescence: Bridging Experiments with Molecular Dynam-
ics Simulations , Springer International Publishing (2016).

[17] J. T. Vivian and P. R. Callis, Biophys. J., 80, 2093 (2001).

[18] G. D. James A. Morris, Russell Martenson and R. H. Cagan, J. Biol. Chem., 248, 534
(1973).

[19] R. Spadaccin, O. Crescenzi, T. Tancred, N. DeCasamassimi, G. Savian, R. Scognamiglio,

A. D. Donato, and P. A.Temussi, J. Mol. Biol., 305, 505 (2001).

[20] L. Nilsson and B. Hallet, Proc. Natl. Acad. Sci., 102, 13867 (2005).

[21] R. Feynman, R. Leighton, and M. Sands, The Feynman Lectures on Physics, Vol. 1,
Addison-Wesley, New York (1963).

9
[22] M. Karplus, Acc. Chem. Res., 35, 321 (2002).

[23] E. Lindahl, in Molecular Dynamics Simulation, edited by A. Kukol, Molecular Modeling of

Proteins Vol. 1215, Humana Press (2006).

[24] F. E. Boas and P. B. Harbury, Curr. Opin. Struct. Biol., 17, 199 (2007).

[25] N. Schmid, A. P. Eichenberger, A. Choutko, S. Riniker, M. Winger, A. E. Mark, and W. F.

Gunsteren, Eur. Biophys. J., 40, 843 (2011).

[26] M. P. Allen and D. J. Tildesley, Computer Simulation of Liquids, Clarendon Press, Oxford
(1989).

Supervisor:

.......................................
Prof. Dr. Narayan Prasad Adhikari
Central Department of Physics
Tribhuvan University
Kathmandu, Nepal

Co-supervisor:

.....................
Dr. Ali Hassanali
The Abdus Salam International Centre for Theoretical Physics
Trieste, Italy

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