Title Dynamics of Fluorescent Probes in Biological
Advisers Prof. N. Periasamy, TIFR, Mumbai, India
Thesis Committee Prof. A. K. Lala, IIT, Mumbai, India; Prof.
G. Govil, Professor, TIFR, Mumbai, India; Prof. S. R. Kasturi, Professor,
TIFR, Mumbai, India; Dr. G. K. Jarori, TIFR, Mumbai, India; Dr. S.
Wategaonkar, TIFR, Mumbai, India
Biological systems are natural complex systems. The
diverse but coordinated functions carried out by these systems are
responsible for the functioning of complex living organisms. Studying
the structure and dynamics of these systems is essential to understand
the physicochemical processes associated with them. The study of biological
systems using the techniques of spectroscopy forms a major discipline
of modern physical chemistry. My thesis deals with the study of dynamics
of small molecules in biological systems, mainly using fluorescence
spectroscopy. The biological systems studied include lipid bilayer
membranes, proteins, and micelles. The main aims of the thesis were
(a) to determine the location, orientation and dynamics of small molecules
in lipid bilayer membranes, (b) to study the refractive index effect
on the fluorescence lifetimes in biological systems and (c) to understand
how a biological/microheterogeneous system affects the photophysics/photochemistry
of small molecules compared to homogeneous solvents.
The location and orientation of small molecules in
lipid bilayer membranes were determined using the effects of viscosity
and refractive index on the fluorescence lifetimes. Increase of
viscosity increases the fluorescence lifetime whereas an increase
in refractive index decreases the fluorescence lifetime. I have studied
these effects in the case of bilayer membranes and proteins in detail.
The refractive index effect is severe in the case of biological systems
compared to homogeneous solvents. The fluorescence lifetime of a small
molecule located in three different sites: surface, interface and
core of the membrane show a characteristic variation with the aqueous
parameters and hence was used to identify the location. The fluorescent
probes located near the surface show a predominant viscosity effect
whereas the effect of refractive index is severe for the probes located
in the interior of the membrane. The two order parameters determined
from fluorescence lifetime and anisotropy measurements do not match.
These were used to show that the small molecules exist in a bimodal
orientational distribution in the interior of the lipid membrane.
These two orthogonal populations do not interconvert on fluorescence
The location and orientation of small molecules in the
membrane depend upon the chain length and unsaturation of the lipid
chain. With an increase of the lipid chain length, the molecules are
pushed more towards the surface whereas with an increase of the acyl
chain unsaturation, more molecules penetrate into the core of the
membrane. More molecules orient parallel to the acyl chains with an
increase of the chain length or with the decrease of the acyl chain
During the course of the above work, a new method of
data analysis, spectrally constrained global analysis (SCGA), was
developed to extract the fluorescence spectra of different components
in a multicomponent system using the known spectra of some of the
components. This was applied to the case of lipid membranes to separate
the aqueous and membrane components.
Coming to the case of dynamics of small molecules,
well documented models such as wobbling-in-a-cone model explain the
experimental results in biological systems. However, no theory exists
for the case of one of the important dynamics in biological systems,
namely translational diffusion on curved surfaces. These equations
are necessary for understanding biological transport phenomena at
a molecular level where the translational diffusion of solutes bound
to different curved surfaces directly influences the rate of metabolism
or the rate at which the chemical signals are conveyed. I developed
a Monte Carlo simulation approach to simulate this diffusion and applied
to the case of fluorophores diffusing on a sphere. Using the principles
of quantum chemistry, the corresponding analytical solutions were
also obtained. In general, the fluorescence anisotropy decay is three
exponential. This study corrects the wrong equations used in the literature.
The same equations also apply in the case of NMR and ESR spin relaxation
measurements. The correct equations were used in interpreting the
fluorescence anisotropy decays in micelles. The orientation of the
molecular dipole was determined. The Monte Carlo simulation approach
developed here will be particularly helpful in the case of complicated
biological curved surfaces where an analytical treatment is not possible.
These methodologies were extended in solving the other problems related
to the surface diffusion . The same simulation and theoretical approaches
were used in understanding the rotational diffusion of surface probes.
The results were applied to the case of sonicated and giant liposomes.
The measured value of anisotropy at infinite time (r¥)
depends on the surface on which the molecules are diffusing and the
way it is measured. This points out the error in using the value of
r¥ as an indication of the dynamical
freedom of the probe.
The refractive index effect was found to be severe
in the case of proteins with buried fluorophores of high quantum yield
(e.g. Green Fluorescent Protein (GFP)), but is not very severe in
the case of tryptophans in proteins because of their low quantum yield
(e.g., Barstar and human seminal plasma prostatic inhibin (HSPI)).
Using the crosspeak patterns in 2D NOESY and COSY NMR spectra, I identified
the two tryptophans in human seminal plasma prostatic inhibin (HSPI)
to exist as single rotamers and hence they show a single fluorescence
lifetime. This result also unequivocally demonstrates the origin of
multiexponential decay of tryptophan in proteins is because of the
multiple rotamers the tryptophan sidechain can adopt. This has been
the first report of its kind in using the NMR results to explain the
fluorescence decay of a multitryptophan protein.
The photophysics/photochemistry of small molecules
in biological and microheterogeneous systems is considerably different
from that in homogeneous solvents. The microheterogeneous media stabilizes
some of the species that are not observable in homogeneous solvents.
The hydrophobic probe Nile red exhibits multi exponential fluorescence
decay with negative amplitudes in membranes and micelles. The observation
of negative amplitudes is not very common. This phenomenon is due
to the excited state kinetics and was attributed to excited state
solvent relaxation. This study points out the fact that the origin
of multiple lifetimes for a fluorescent probe in a biological system
may not be from different species located at different sites but can
also be due to the probe located at a single site and undergoing excited
state kinetics. Red edge excitation shifts (REES) was observed in
the case of Nile red - hydrophobic protein complexes indicating the
ground state heterogeneity. Methods were developed to distinguish
between two state and continuous excited state solvent relaxation
. In the case of voltage sensitive aminostyryl pyridinium dyes, a
stable fluorescent quinoid state was observed in viscous, microheterogeneous
media and in halosolvents. In the case of fluorescence dynamics in
micelles, nonbrownian dynamics was observed that do not follow Stokes-Einstein
I observed new fluorescent photoproducts with red shifted
emission and long fluorescence lifetimes in the case of biological
fluorophores: indole and its derivatives, tryptophan and melatonin.
Mercury quenching and tryptophan photobleaching were used to identify
the Mercury binding sites in aquaporins (AQP1) that inhibit their
water channel function. Using these two techniques, methods were developed
to selectively silence one of the tryptophans in a multitryptophan
My current postdoctoral research is focused on the chaperonin
assisted protein folding problem in understanding these complicated
biological machines that maintain our life. The award of this IUPAC
Prize for Young Chemists will surely help me and encourage me in establishing
my future research career in the area of biophysical chemistry.