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IUPAC Prize for Young Chemists - 2000
Honorable Mention

 

 

Mallela M.G. Krishna receives one of five Honorable Mention awards associated with the IUPAC Prize for Young Chemists, for his Ph.D. thesis work entitled "Dynamics of Fluorescent Probes in Biological Systems"

Current address (at the time of application)

Department of Biochemistry and Biophysics
University of Pennsylvania
School of Medicine
Philadelphia, PA 19104-6059, USA

Tel.: +1 215 898 4509/6580
Fax: +1 215 898 2415
E-mail: kmallela@mail.med.upenn.edu

Academic degrees

  • Ph.D. in Physical Chemistry, May 1999, Department of Chemical Sciences, Tata Institute of Fundamental Research (TIFR), University of Mumbai, Mumbai, India.
  • M. Sc. in Chemistry, April 1993, School of Chemistry, Central University of Hyderabad, Hyderabad, India.
  • B. Sc. in Chemistry, Mathematics and Physics, May 1991, P. B. Siddhartha Autonomous College of Arts and Science, Nagarjuna University, AP, India.

Ph.D. Thesis

Title Dynamics of Fluorescent Probes in Biological Systems
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

Essay

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 timescale.

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 unsaturation.

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 relations.

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 protein.

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.


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