Scientist, IR spectroscopist, Ligand binding

Curriculum Vitae

Name: Saroj Kumar

Sex: Male

Professional address: Laboratory for Structure and Function of Biological Membranes

Universite Libre de Bruxelles, Campus Plaine, Building BC, Boulevard

duTriomphe, 1050 Brussels, Belgium, +32-471******,

acbk87@r.postjobfree.com

Career Objective

A challenging position requiring the innovation, hard work, consultancy and expertise

attained in field of science.

Education, main scientific appointments

Postdoctoral fellow (Dec.2011-present) Laboratory for Structure and Function of Biological

Membranes, Université Libre de Bruxelles, Brussels, Belgium

Histology of breast cancer improved by infrared imaging

Research assistant (Aug-Nov.2011), Department of Biochemistry and Biophysics,

Stockholm University, Sweden

PhD (2011) Department of Biochemistry and Biophysics, Stockholm University, Sweden,

Advisor: Professor Andreas Barth

Thesis: “Infrared spectroscopy: Method development and ligand binding to pyruvate

kinase”

Side projects:

Energy coupling in the Ca2+-ATPase (mainly expression and purification of

recombinant ATPase) and H+, K+-ATPase studied by Fourier transform IR

(FTIR) spectroscopy

Study of yeast and bacterial cells by attenuated total reflection (ATR)-FTIR

spectroscopy

Licentiate degree (2010) Department of Biochemistry and Biophysics, Stockholm

University, Sweden

Advisor: Professor Andreas Barth

Thesis: “Infrared spectroscopy: Method development and ligand binding studies”

Degree project (2006) Department of Biochemistry and Biophysics, Stockholm University,

Sweden

Redox linked conformational changes in cytochrome c and Protein-herbicide

interaction studied by ATR-FTIR spectroscopy

Indian council of medical research fellow (2004) Institute of pathology ICMR, Safdarjung,

New Delhi, India

Microscopic colitis studied by electron microscopy

Master of Science in Biophysics (2004) All India institute of medical sciences, New Delhi,

India

Extensive course work in theoretical and molecular biophysics

Master Thesis: “Cloning of cobra venom phospholipase A2”

Bachelor of Science in human biology (2001) All India institute of medical sciences, New

Delhi, India

anatomy, biochemistry, biophysics, pathology, microbiology, physical and

chemical sciences

Professional activities

20% teaching duty of PhD (since 2007) and supervision of graduate and postgraduate

students

Supervising high school students during summer training (since 2008)

Attended 5 workshops and 16 conferences on spectroscopy and structural proteomics

Collaboration with a large chemical company (2005): plant herbicide study by ATR-

FTIR

PhD and postdoctoral scholarships

Brains back to Brussels (2011) to pursue postdoc (only in exceptional cases given to

people not coming from Belgium)

Postdoctoral grant from National fund for scientific research, Belgium (2011)

Swedish postdoctoral grant (2011) to pursue postdoc studies

Marie Curie International outgoing fellowship for career development (2011),

proposal was placed on the reserve list

Lawski fellowship (2006-2009) to pursue PhD study

Other scholarships and awards

W. Bagge och Rhodin donationstipendium (2009): 1400 Euro project grant

K & A Wallenbergs stiftelse (2008) travel grant to participate in the 5th International

Conference on Advanced Vibrational Spectroscopy, Melbourne, Australia

C. F. Liljevalch J:ors (2008) travel grant to participate in 2nd international workshop on

expression, structure and function of membrane Proteins, Florence, Italy

Techniques worked with

Time resolved Fourier transform infrared spectroscopy (FTIR)

FTIR microspectroscopy

Attenuated total reflection FTIR spectroscopy

UV-visible spectroscopy

Circular dichroism spectroscopy

Fluorescence spectroscopy

X-Ray crystallography

Eukaryotic cell culture

Cell culture of human cell lines

Expression and purification of membrane proteins (Ca2+-ATPase)

Cloning, sequencing, polymerase chain reaction (PCR)

Electrophoresis, Western blot

Monolayer preparation of lipids

Electron microscopy

Crystallization

Histopathology slide preparation and microscopical studies

Computation skills

Application software related to processing of scientific data, MS office, Origin, Pymol, and

Kinemage, Matlab based program Kinetics

Research results

Publications/Manuscripts

S.Kumar, C.Desmedt, D.Larsimont, C.Sotiriou, E.Goormaghtigh: Change in the

microenvironment of breast cancer studied by FTIR imaging. Analyst, 2013, 138,

4058-65

S.Kumar, C.Li and A.Barth: Conformational changes of recombinant Ca2+-ATPase

studied by reaction-induced infrared difference spectroscopy. FEBS J. 2013, 13 Feb,

1-10

A.Valdivia, A.Barth, Y.Batista and S.Kumar: Characterization of recombinant

antibodies for cancer therapy by infrared spectroscopy. Biologicals, 2013, 1-7

S.Kumar, N.Eremina and A.Barth: Detection of Ligand Binding to Proteins through

Observation of Hydration Water. J. Phys. Chem. B. 2012, 116, 13968-74

S.Kumar and A.Barth: Allosteric effects of fructose bisphosphate on muscle pyruvate

kinase studied by infrared spectroscopy. J. Phys. Chem. B. 2011, 115 (39), 11501-05

S. Kumar and A. Barth: Effects of ions on ligand binding to pyruvate kinase: mapping

the binding site with infrared spectroscopy. J. Phys. Chem. B. 2011, 115, 6784-89

S.Kumar and A.Barth: Following enzyme activity with infrared spectroscopy. Sensors

2010, 10, 2626-37

S.Kumar and A.Barth: Phosphoenolpyruvate and Mg2+ Binding to Pyruvate Kinase

Monitored by Infrared Spectroscopy. Biophys. J., 2010, 98, 1931-40

M.Rudbeck, S.Kumar, M. Blomberg, M-A. Mroginski, A. Barth: Infrared spectrum of

phosphoenol pyruvate: computational and experimental studies. J. Phys. Chem. A.

2009, 113, 2935-42

M.Krasteva, S.Kumar and A.Barth: A dialysis accessory for attenuated total reflection

IR spectroscopy. Spectroscopy (cover page) 2006, 20, 89-94

T.S.Shabi, S.Kumar, E. Goormaghtigh, Y. Geerts: Role of solubilizing group removal

rate on the grain size and crystallinity of diketopyrrolopyrrole based compounds and

its impact on their optical and electrical properties. Crystal growth and design,

Accepted

C.Li, S.Kumar and A.Barth: Effect of E309Q on Ca2+-ATPase pump studied by

reaction-induced Infrared difference spectroscopy. Manuscript in preparation

S.Singh, S.Kumar, A.Barth and M. Malmsten: Membrane and lipopolysaccharide

interactions of C-terminal peptides from S1 peptidases by infrared spectroscopy.

Manuscript in preparation

Reviews/Books

S.Kumar: Infrared spectroscopy is a physical tool to study biomolecules. Applied

spectroscopy reviews, 49 (3), 2014, 187-200 (online)

S.Kumar: Infrared spectroscopy: Method development and ligand binding to pyruvate

kinase. 2011,, pp 1-57, ISBN: ***-**-****-297-4

Reviewer for scientific journals

Spectroscopy letters

Environmental science and technology

Molecular pharmaceutics

International Conferences/Workshops (International)

1. S.Kumar and E. Goormaghtigh (2012): Conference Spec 2012, “Shedding new light

on disease”. Chaing Mai, Thailand

2. S.Kumar, and A.Barth (2009): 2nd international workshop on expression, structure and

function of membrane proteins. Poster presentation and actively participate in

workshop. Florence, Italy

3. S.Kumar and A.Barth (2009): Infrared studies of ligand binding and enzyme activity

of biomoleules. Poster presentation at 5th International conference on advanced

vibrational spectroscopy, Melbourne, Australia

4. S.Kumar, and A.Barth (2008): Ligand induced conformational changes of pyruvate

kinase study by ATR-FTIR spectroscopy. Poster presentation at XXIX European

conference on molecular spectroscopy, Opatija, Croatia

5. S.Kumar (2008): Workshop in protein production for structural genomics, at Structural

genomics consortium (SGC), Stockholm

6. S.Kumar, and A.Barth (2007): Protein-ligand interaction study by ATR-FTIR

spectroscopy. Poster presentation at 12th European conference on the spectroscopy of

biological molecules, Paris, France

7. S.Kumar, M.Krasteva and A.Barth (2005): Protein-ligand interaction study by ATR-

FTIR spectroscopy. Poster presentation at 11th European conference on the

spectroscopy of biological molecules, Aschaffenburg, Germany

Oral presentations (External)

1. 2013: 15th European Conference on the Spectroscopy of Biological Molecules,

Oxford, UK

2. 2010: CEA, Saclay, Gif sur Yvette, France

3. 2010: 30th European Congress on Molecular Spectroscopy. Florence, Italy

Major accomplishments in research

Method development for infrared spectroscopy

I have taken part in successfully developing the three methods for infrared spectroscopy. The

first method couples a dialysis accessory to an attenuated total reflection unit to study ligand

binding to macromolecules. The main advantage of this method is the possibility to

manipulate the sample of interest during an experiment and to perform multiple experiments

with different ligands on the same sample which saves time and material. This work has been

published (#10). We developed a second method for infrared spectroscopy which provides a

direct, monitor of enzymatic reactions of three enzymes. The main advantage of the infrared

method is that it observes the reaction of interest directly, i.e. no activity assay is required that

converts the progress of the reaction into an observable quantity as often necessary for

spectroscopic detection in the UV/visible spectral range. This work has been published in

(#7). The third method is a novel approach to detect ligand binding by a change in water

absorption. The idea behind this work was that water is displaced in all ligand binding process

from the hydration shell of proteins and ligands to bulk water. Infrared spectroscopy has a

high sensitivity for water interactions and any changes in the hydrogen bond pattern can be

detected. Using the absorption of the OH stretching mode we were able to detect these

changes. This work has been published (#4).

The molecular mechanism of pyruvate kinase (PK)

PK is an important enzyme of the glycolytic pathway that catalyses the transfer of phosphate

from phosphoenolpyruvate (PEP) to adenosine diphosphate. By using infrared spectroscopy,

we observed that all types of secondary structures are affected upon PEP binding to PK. We

observed that the enzymatic environment affected the structure of the bound substrate, but

none of the bonds of PEP were significantly distorted upon binding, in particular not those of

the reacting phosphate group. We also studied the effects of monovalent and divalent cations

on binding of the substrate PEP to PK, as both types of cations are necessary for PK's activity.

We observed that the binding mode of PEP is affected more by different divalent cations than

by different monovalent cations, which shows that the divalent cation plays a more decisive

role for the structural consequences of PEP binding. Mn2+ showed very interesting effects as

PEP binding produced larger signals than with the physiological ion Mg2+, indicating a more

closed conformation after PEP binding. Furthermore, we studied the effects of the allosteric

effector fructose 1, 6 bisphosphate (FBP) on PEP binding to PK in presence of different ion

combinations. The results indicate that FBP binding shifts the equilibrium of the

PK:PEP:Mg2+:K+ complex towards the closed conformation. The PEP concentration for

saturating signal was lower with FBP than without which shows that the affinity of PK for

PEP increased by binding of FBP. This work has resulted in four publications (#5, 6, 8, 9). My

contribution was the planning and execution of the project.

Energy coupling in the Ca2+-ATPase

The Ca2+-ATPase is a P-type ATPase which pumps two calcium ions across the sarcoplasmic

reticulum membrane at the expense of the hydrolysis of one ATP molecule. The aim of the

project was to understand how chemical energy from ATP is converted to directional Ca2+

transport by the Ca2+-ATPase by studying the effects of amino acid substitutions. As an

essential first step, I established in our laboratory the expression and purification system of

recombinant Ca2+-ATPase and obtained active protein. It was characterized by biophysical

methods and compared with the native Ca2+-ATPase. This work represents the majority of my

work during my PhD project. It was done in the settings of a biophysics group without

previous experience in molecular biology and initially without appropriate equipment. This

work has resulted in one publication (#2) and a manuscript of it is in preparation. My

contribution was the independent establishing of the expression and purification in our

laboratory from a published protocol.

Histology of breast cancer improved by infrared imaging

Breast cancer is a global public health issue. At diagnosis, 90% of the patients appear to have

operable breast cancer, that is, the disease that is confined to the breast and ipsilateral axilla.

However, only up to 40% are cured by the operation while around 50% of these women will

develop metastatic disease (incurable). Although different tools have been developed to assist

clinicians in selecting patients who should receive adjuvant therapy, it still remains a

challenge to distinguish those patients who would really need adjuvant systemic therapy from

those who could be spared such a treatment. Fourier transform infrared (FTIR) imaging is a

suitable technique for the quantitative analysis of tissue in the clinical routine. This

quantitative analysis should allow a description of the most common cell types found on

histological slices and a grading of tumor tissues from microscopic observation of the slices,

presence of markers and clinical observations. Particular attention is paid to the influence of

the microenvironment (stroma) on cancer progression. I found a spectral marker which

distinguishes the infected and normal stroma which resulted a publication (#1).

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