Study of Biomolecular Recognition Using Time-Resolved Optical Spectroscopy

Tanumoy Mondol(1*), Soma Banerjee(2), Subrata Batabyal(3), Samir Kumar Pal(4)

(1) Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India, India
(2) Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India, India
(3) Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India, India
(4) Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India, India
(*) Corresponding author

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Molecular recognition process refers to the weak non-covalent interaction, which takes place selectively and specifically between small ligand molecules with biological macromolecules. Understanding of such recognition in biological and biomimetic milieu is the central attraction for drug designing, which is crucial for the improvement of human healthcare. A thorough knowledge of the structural, dynamical and energetic parameters that dictate such molecular interactions can find immense use in the modulations of the ligand-macromolecule recognition process. In this article, we present our continuous effort to investigate the fundamental physical processes involved in the biomolecular recognition, e.g. efficiency (binding affinity and rigidity of the complex) and role of solvent molecules in the molecular recognition using steady state and predominantly, ultrafast time-resolved fluorescence spectroscopy. In this perspective, we have thoroughly investigated the molecular recognition of small ligand/drug molecules (Rifampicin; Rf, 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran; DCM, and Nile Blue; NB) by a human transporter protein, Human Serum Albumin (HSA), and also established the nonspecific type of interaction between a ligand molecule (Rf) and a biomimetic system (Sodium Dodecyl Sulfate (SDS) micelle). Simultaneous recognition of an intercalator (Ethidium Bromide, EtBr) and a DNA minor groove binder (Hoeschst 33258, H258) to a dodecamer DNA of specific sequence has also been monitored. Besides, we report an investigation on the recognition of ethidium (Et) cation, a potential mutagen, by synthetic DNA and various cell nuclei in presence of a stimulant drug, caffeine, employing the mentioned spectroscopic techniques along with NMR and fluorescence microscopy. Moreover, we have explored the differential molecular recognition of 8-anilino-1-naphthalenesulfonic acid (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS) by bovine pancreatic -chymotrypsin (CHT) upon interaction with genomic DNA. The correlation of the molecular recognition of the DNA and DNA-protein complexes with the hydration dynamics has been further exploited in our studies. In addition, we have developed functional nanoparticles/Quantum dots (QDs) that are covalently linked to biological molecules to detect the molecular interaction phenomenon between biomolecules. It should be noted that QDs have a significant contribution in the field of nano-biotechnology due to its high quantum yield, low photo-bleaching and increased biological application (cell labeling, in vivo imaging, gene delivery, sensing of fluorescence and molecular recognition). In this regard, we have exploited QDs as a potential energy donor/acceptor system and validated Förster resonance energy transfer (FRET) model over nano-surface energy transfer (NSET) technique. Therefore, the ultrafast non-radiative energy migration from tryptophan (Trp214) present in HSA to the HSA bound CdS QD, and from 4-nitrophenyl anthranilate (NPA) to Silver (Ag) nanoclusters in CHT (both NPA and Ag bound to CHT) have been investigated using FRET technique to monitor the protein folding pathway of HSA, and molecular interaction between NPA and CHT respectively. Moreover, we have also used functionalized QDs (CdSe/ZnS) for the detection of molecular recognition of ethidium bromide (EtBr) by a synthetic DNA. However, the intention of this review is to give an overview of ultrafast optical spectroscopic techniques for the exploration of biomolecular recognition, which may find potential significance for further research in the field of nano-biotechnology and medicine
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Biomolecular Recognition; Ultrafast Spectroscopy; NMR Spectroscopy; Förster Resonance Energy Transfer (FRET)

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X. Luo, D. Zhang, et al., Ligand-induced domain motion in the activation mechanism of a G-protein-coupled receptor, Protein Eng., volume 7, (issue 12), December 1994, pages 1441-1448.

J.R. Warner, R. Soeiro, The Involvement of RNA in Protein Synthesis, N. Engl. J. Med., volume 276, (issue 10), March 1967, pages 563-570.

J.M. Gottesfeld, L. Neely, et al., Regulation of gene expression by small molecules, Nature, volume 387, (issue 6629), May 1997, pages 202-205.

D.A. Koster, K. Palle, et al., Antitumour drugs impede DNA uncoiling by topoisomerase I, Nature, volume 448, (issue 7150), July 2007, pages 213-217.

C.R. Kahl, A.R. Means, Regulation of Cell Cycle Progression by Calcium/Calmodulin-Dependent Pathways, Endocr. Rev., volume 24, (issue 6), December 2003, pages 719-736.

D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry (W. H. Freeman & Co., 2000).

S.H. Gellman, Introduction: Molecular Recognition, Chem. Rev., volume 97, (issue 5), August 1997, pages 1231-1232.

G.M. Verkhivker, P.A. Rejto, et al., Towards understanding the mechanisms of molecular recognition by computer simulations of ligand–protein interactions, J. Mol. Recognit., volume 12, (issue 6), December 1999, pages 371-389.

S. Banerjee, D. Bhowmik, et al., Ultrafast Spectroscopic Study on Caffeine Mediated Dissociation of Mutagenic Ethidium from Synthetic DNA and Various Cell Nuclei, J. Phys. Chem. B, volume 115, (issue 49), December 2011, pages 14776-14783.

D. Banerjee, P.K. Verma, et al., Temperature-dependent femtosecond-resolved hydration dynamics of water in aqueous guanidinium hydrochloride solution, Photochem. Photobiol. Sci, volume 8, (issue 10), July 2009, pages 1441-1447.

R. Sarkar, M. Ghosh, et al., Ultrafast relaxation dynamics of a biologically relevant probe dansyl at the micellar surface, J. Photochem. Photobiol., B, volume 78, (issue 2), February 2005, pages 93-98.

D.V. O'Connor, D. Phillips, Time-Correlated Single Photon Counting (Academic Press, 1984).

J.R. Lakowicz, Principles of Flourescence Spectroscopy (Kluwer Academic/Plenum Publishers, 1999).

P. Majumder, R. Sarkar, et al., Ultrafast dynamics in a nanocage of enzymes: Solvation and fluorescence resonance energy transfer in reverse micelles, J. Colloid Interface Sci., volume 290, (issue 2), October 2005, pages 462-474.

M. Montalti, N. Zaccheroni, et al., Enhanced Sensitized NIR Luminescence from Gold Nanoparticles via Energy Transfer from Surface-Bound Fluorophores, J. Am. Chem. Soc., volume 129, (issue 9), March 2007, pages 2418-2419.

B.N.J. Persson, N.D. Lang, Electron-hole-pair quenching of excited states near a metal, Phys. Rev. B, volume 26, (issue 10), November 1982, pages 5409-5415.

D. Craig, T. Thirunamachandra, Molecular quantum Electrodynamics (Academic Press, 1984).

M.A.H. Muhammed, A.K. Shaw, et al., Quantum Clusters of Gold Exhibiting FRET, J. Phys. Chem. C, volume 112, (issue 37), September 2008, pages 14324-14330.

S. Batabyal, A. Makhal, et al., Ultrafast dynamics of excitons in semiconductor quantum dots on a plasmonically active nano-structured silver film Nanotechnology, volume 22, (issue 19), May 2011, pages 195704-195707.

A. Makhal, H. Yan, et al., Light Harvesting Semiconductor Core−Shell Nanocrystals: Ultrafast Charge Transport Dynamics of CdSe−ZnS Quantum Dots, J. Phys. Chem. C, volume 114, (issue 1), December 2009, pages 627-632.

C. Burda, S. Link, et al., The Relaxation Pathways of CdSe Nanoparticles Monitored with Femtosecond Time-Resolution from the Visible to the IR: Assignment of the Transient Features by Carrier Quenching, J. Phys. Chem. B, volume 105, (issue 49), November 2001, pages 12286-12292.

S. Shankara Narayanan, S.S. Sinha, et al., Ultrafast energy transfer from 3-mercaptopropionic acid-capped CdSe/ZnS QDs to dye-labelled DNA, Chem. Phys. Lett., volume 463, (issue 1-3), September 2008, pages 160-165.

B.A. Korgel, Nanoparticle assemblies: Interfaces behaving well, Nat. Mater., volume 6, (issue 8), August 2007, pages 551-552.

T. Mondol, P. Rajdev, et al., Interaction of an Antituberculosis Drug with a Nanoscopic Macromolecular Assembly: Temperature-Dependent Förster Resonance Energy Transfer Studies on Rifampicin in an Anionic Sodium Dodecyl Sulfate Micelle, J. Phys. Chem. B, volume 115, (issue 12), March 2011, pages 2924-2930.

P. Rajdev, T. Mondol, et al., Simultaneous binding of anti-tuberculosis and anti-thrombosis drugs to a human transporter protein: A FRET study, J. Photochem. Photobiol., B, volume 103, (issue 2), May 2011, pages 153-158.

S.S. Sinha, R.K. Mitra, et al., Temperature-Dependent Simultaneous Ligand Binding in Human Serum Albumin, J. Phys. Chem. B, volume 112, (issue 16), April 2008, pages 4884-4891.

D. Banerjee, S.K. Pal, Simultaneous Binding of Minor Groove Binder and Intercalator to Dodecamer DNA: Importance of Relative Orientation of Donor and Acceptor in FRET, J. Phys. Chem. B, volume 111, (issue 19), May 2007, pages 5047-5052.

R. Langer, Drugs on Target, Science, volume 293, (issue 5527), July 2001, pages 58-59.

J. Huwyler, D. Wu, et al., Brain drug delivery of small molecules using immunoliposomes, Proc. Natl. Acad. Sci., volume 93, (issue 24), November 1996, pages 14164-14169.

J.A. Hubbell, Enhancing Drug Function, Science, volume 300, (issue 5619), April 2003, pages 595-596.

M.A. Hillmyer, Micelles Made to Order, Science, volume 317, (issue 5838), August 2007, pages 604-605.

S.M. Sullivan, L. Huang, Enhanced delivery to target cells by heat-sensitive immunoliposomes Proc. Natl. Acad. Sci., volume 83, (issue 16), August 1986, pages 6117-6121.

D. Banerjee, S.K. Pal, Ultrafast charge transfer and solvation of DNA minor groove binder: Hoechst 33258 in restricted environments, Chem. Phys. Lett., volume 432, (issue 1-3), 2006, pages 257–262.

D.A. Mitchison, The Garrod Lecture. Understanding the chemotherapy of tuberculosis – current problems, J. Anitimicrob. Chemother., volume 29, (issue 5), May 1992, pages 477-493.

T.H. Self, R.B. Mann, Interaction of Rifampicin and Warfarin, Chest, volume 67, (issue 4), April 1975, pages 490-491.

W. Kyoichi, K. Etsuko, et al., Interaction of Warfarin and Rifampicin on Medical Report, Jpn. J. Pharm. Hlth. Care Sci., volume 28, (issue 1), 2002, pages 85-90.

R. Wetzel, M. Becker, et al., Temperature behaviour of human serum albumin, Eur. J. Biochem., volume 104, (issue 2), March 1980 pages 469-478.

K. Flora, J.D. Brennan, et al., Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods, Biophys. J., volume 75, (issue 2), August 1998, pages 1084-1096.

G. Bohm, R. Muhr, et al., Quantitative analysis of protein far UV circular dichroism spectra by neural networks, Protein Eng., volume 5, (issue 3), April 1992, pages 191-195.

J.D. Yang, S.X. Deng, et al., Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application, Luminescence, volume 22, (issue 6), Nov-Dec 2007, pages 559-566.

C.E. Petersen, C.E. Ha, et al., Probing structure of the warfarin binding site on human serum albumin using site directed mutagenesis, Proteins: Struct., Funct., Genet., volume 47, (issue 2), May 2002, pages 116-125.

I. Petitpas, A.A. Bhattacharya, et al., Crystal Structure Analysis of Warfarin Binding to Human Serum Albumin, J. Biol. Chem., volume 276, (issue 25), April 2001, pages 22804-22809.

S. Patel, A. Datta, Steady State and Time-resolved Fluorescence Investigation of the Specific Binding of Two Chlorin Derivatives with Human Serum Albumin, J. Phys. Chem. B, volume 111, (issue 35), August 2007, pages 10557-10562.

S.S. Krishnakumar, D. Panda, Spatial Relationship between the Prodan Site, Trp-214, and Cys-34 Residues in Human Serum Albumin and Loss of Structure through Incremental Unfolding, Biochemistry, volume 41, (issue 23), June 2002, pages 7443-7452.

S.K. Pal, D. Mandal, et al., Solvation Dynamics of DCM in Human Serum Albumin, J. Phys. Chem. B, volume 105, (issue 7), February 2001, pages 1438-1441.

R. Sarkar, S.K. Pal, Ligand–DNA interaction in a nanocage of reverse micelle, Biopolymers, volume 83, (issue 6), December 2006, pages 675-686.

S.-I. Murata, J. Kuśba, et al., Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations, Biopolymers, volume 57, (issue 5), July 2000, pages 306-315.

P.E. Pjura, K. Grzeskowiak, et al., Binding of Hoechst 33258 to the minor groove of B-DNA, J. Mol. Biol., volume 197, (issue 2), September 1987, pages 257-271.

M. Levitt, How many base-pairs per turn does DNA have in solution and in chromatin? Some theoretical calculations, Proc. Natl. Acad. Sci., volume 75, (issue 2), February 1978, pages 640-644.

S.S. Narayanan, S.K. Pal, Nonspecific Protein−DNA Interactions: Complexation of α-Chymotrypsin with a Genomic DNA, Langmuir, volume 23, (issue 12), May 2007, pages 6712-6718.

D. Banerjee, A. Makhal, et al., Sequence Dependent Femtosecond-Resolved Hydration Dynamics in the Minor Groove of DNA and Histone—DNA Complexes, J. Fluorescence, volume 19, (issue 6), June 2009, pages 1111-1118.

S. Banerjee, P. Verma, et al., Probing the Interior of Self-Assembled Caffeine Dimer at Various Temperatures, J. Fluorescence, DOI: 10.1007/s10895-011-1011-3.

R. Jimenez, G.R. Fleming, et al., Femtosecond solvation dynamics of water, Nature, volume 369, (issue 6480), June 1994, pages 471-473.

R.E. Riter, D.M. Willard, et al., Water Immobilization at Surfactant Interfaces in Reverse Micelles, J. Phys. Chem. B, volume 102, (issue 15), March 1998, pages 2705-2714.

E.M. Corbeil, N.E. Levinger, Dynamics of Polar Solvation in Quaternary Microemulsions, Langmuir, volume 19, (issue 18), July 2003, pages 7264-7270.

K. Hirose, A Practical Guide for the Determination of Binding Constants, J. Incl. Phenom. Macrocyclic Chem., volume 39, (issue 3-4), April 2001, pages 193-209.

L.D. Weber, A. Tulinsky, et al., Expression of functionality of alpha-chymotrypsin. The structure of a fluorescent probe-alpha-chymotrypsin complex and the nature of its pH dependence, Biochemistry, volume 18, (issue 7), April 1979, pages 1297-1303.

D.C. Turner, L. Brand, Quantitative estimation of protein binding site polarity. Fluorescence of N-arylaminonaphthalenesulfonates, Biochemistry, volume 7, (issue 10), October 1968, pages 3381-3390.

A.K. Shaw, R. Sarkar, et al., Direct observation of protein residue solvation dynamics, J. Photochem. Photobiol., A, volume 185, (issue 1), January 2007, pages 76-85.

D. Zhong, S.K. Pal, et al., Femtosecond Studies of Protein–DNA Binding and Dynamics: Histone I, Chemphyschem, volume 2, (issue 4), April 2001, pages 219-227.

S.K. Pal, L. Zhao, et al., Water at DNA surfaces: Ultrafast dynamics in minor groove recognition, Proc. Natl. Acad. Sci. USA, volume 100, (issue 14), June 2003, pages 8113–8118.

R. Sarkar, S.S. Narayanan, et al., Direct Conjugation of Semiconductor Nanocrystals to a Globular Protein to Study Protein-Folding Intermediates, J. Phys. Chem. B, volume 111, (issue 42), October 2007, pages 12294-12298.

S.S. Narayanan, S.K. Pal, Structural and Functional Characterization of Luminescent Silver−Protein Nanobioconjugates, J. Phys. Chem. C, volume 112, (issue 13), March 2008, pages 4874-4879.

R.P. Haugland, L. Stryer, in Conformation of Biopolymers (Academic press, 1967).

D.V. Leff, L. Brandt, et al., Synthesis and Characterization of Hydrophobic, Organically-Soluble Gold Nanocrystals Functionalized with Primary Amines, Langmuir, volume 12, (issue 20), October 1996, pages 4723-4730.


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