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  • 1. Eur. Phys. J. D (2010)DOI: 10.1140/epjd/e2010-00107-7THE EUROPEAN PHYSICAL JOURNAL D Regular ArticleUltrafast resonance energy transfer in bio-molecular systemsP.K. Verma and S.K. PalaUnit for Nano Science & Technology, Department of Chemical, Biological & Macromolecular Sciences,S.N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, 700098 Kolkata, India Received 14 December 2009 / Received in nal form 28 January 2010 Published online 26 April 2010 c EDP Sciences, Societ` Italiana di Fisica, Springer-Verlag 2010a Abstract. In this article, we present our consistent eorts to explore the dynamical pathways of the mi- gration of electronic radiation by using ultrafast (picosecond/femtosecond time scales) Frster resonanceo energy transfer (FRET) technique. The ultrafast non-radiative energy migration from an intrinsic donor uorophore (Tryptophan, Trp214) present in domain IIA of a transporter protein human serum albumin (HSA) to various non-covalently/covalently attached organic/inorganic chromophores including photopor- phyrin IX (PPIX), polyoxovanadate [V15 As6 O42 (H2 O)]6 clusters (denoted as V15 ) and CdS quantum dots (QDs) has been explored. We have also used other covalently/non-covalently attached extrinsic uo- rogenic donors (NPA, ANS) in order to exploit the dynamics of resonance energy migration of an enzyme -chymotrypsin (CHT). The use of extrinsic donor instead of intrinsic Trp in CHT avoids ambiguity in the location of the donor molecule as seven tryptophans are present in the enzyme CHT. We have labeled CHT with ANS (1-anilinonaphthalene-8-sulfonate) and NPA (4-nitrophenyl anthranilate) and studied FRET. Labeling of DNA has also been done in the context that the DNA bases have very low quantum yield for uorescence. We have also validated FRET model over nano-surface energy transfer technique (NSET) in the case of quantum clusters and applied the ndings to other QDs. The use of QDs over organic uorophore was justied by least photo-bleaching of QDs compared to organic uorophore. Our studies may nd relevance in the exploration of alternate pathway for ultrafast migration of electronic radiation through FRET to minimize the detrimental eect of UV radiation in living organism.1 Introduction strand [7]. DNA dimerization of pyrimidines induced by UV leads to the distortion of DNA structure, which maySunlight, which is essential for life on earth, contains sig-result in cytotoxicity, mutagenicity, and the induction ofnicant amounts of harmful UV ( < 400 nm) radiation.cell signaling pathways. Indeed, pyrimidine dimers canThese solar UV photons constitute one of the most ubiq-block DNA replication, cell division, and DNA transcrip-uitous and potent environmental carcinogens. Many stud-tion needed for the synthesis of messenger RNA, which is aies have demonstrated that ultraviolet (UV) radiation cancrucial step for protein production and cell survival. Thesedamage proteins or cell membranes, and generate oxida- considerations explain why cells have evolved elaboratetive stress, it is generally accepted that its major delete- mechanisms to identify and repair damage before it blocksrious eects are mediated by specic DNA lesions arising replication or causes a mutation. Cells would not endurefrom the direct absorption of photons. These lesions havelong without such mechanisms. A process called photore-been reported to play an important role in the induction activation uses DNA photolyase to directly reverse theof the lethal, mutagenic, and tumorigenic eects of UV formation of pyrimidine dimers that results from UV ir-exposure [1]. Ultraviolet radiation poses, in this respect,radiation [8,9]. Photolyases are widespread in nature and,a serious threat to the well-ordered sequence of hydrogen- for example, have been reported in many bacteria, blue-bonded DNA base pairs [26]. Although all nucleic acid green algae, fungi, higher plants and all major groupsbases can exist in a variety of energetically close-lying tau- of vertebrates, with the possible exception of placentaltomeric forms, only one tautomer of each species is presentmammals. In photoreactivation, the enzyme DNA pho-in the canonical base pairs of healthy DNA. Knowledge of tolyase captures energy from light and uses it to breakthe photophysical properties of nucleic acid bases is thethe covalent bond linking adjacent pyrimidines [9]. Pho-key to understand radiation induced genetic damage.tolyase is a monomeric protein that contains two chro-UV light was experimentally demonstrated to causemophore/cofactors, avin and folate (or deazaavin). OneDNA damage mostly by the formation of dimeric photo- chromophore (folate) is used for light absorption, and theproducts between adjacent pyrimidine bases on the same other (avin) for chemistry. This is supported by the absorptivities of the two chromophores: folate exhibits a e-mail: = 25 000 M1 cm1 vs. = 5000 M1 cm1 for FADH .

2. 2 The European Physical Journal DIt has been shown that Frster resonance energy transferoportant to establish the validation of FRET over nano-(FRET) occurs via dipole-dipole coupling between folatesurface energy transfer technique (NSET) which comesand avin [10].from the damping of the donor uorophores oscillating dipole by the acceptor QDs through either electron holeThus it is evident that ultrafast migration of electronic pair (EHP) for semiconductor or free electrons for metal.radiation (UV) in DNA is not only important for the pro- In this respect we will discuss the validation of FRET overtection of the genetic material from UV damage but also NSET in the case of quantum metal clusters and apply theequally important for the recovery of the damage through nding to other systems like QDs.a directed resonance energy transfer in biologically rele-vant chromophores. As photolysases are possibly absentin placental mammals it becomes extremely important tond an alternate path through FRET to minimize the 2 Methodologyeect in placental mammals. The ultrafast migration of 2.1 Frster resonance energy transfer (FRET)oelectronic radiation is equally important in the light har-techniquevesting processes in important biological systems includ-ing photosynthesis. In biological systems, light harvestingFRET is an electrodynamic phenomenon involving theuses the energy in photons to drive chemical reactions.non-radiative transfer of the excited state energy fromIn proteins, a series of chromophores are used to separate the donor dipole (D) to an acceptor dipole (A) in thecharges and avoid back reactions. The energy passes be-ground state in a distance dependent manner (Fig. 1a).tween chromophores through FRET. If the chromophores Since the energy transfer eciency is distance dependent,are close enough together typically within a few nanome- it is also known as spectroscopic ruler [15]. FRET is verytres then they are coupled like two pendulums attached tooften used to measure the distance between two sites on athe same beam: if one starts oscillating, it can set the other macromolecule. FRET has got wide uses in all uorescencemoving. This coupling depends not only on distance but applications including medical diagnostics, DNA analy-also on the relative orientation of the two chromophores sis, imaging, sensing, detection, therapy, and monitoring(donor and acceptor). Hence it becomes extremely impor-structural changes of biomolecules. Basically, FRET istant to study FRET in biological systems. Explorationof two types: (a) homo-molecular FRET and (b) hetero-of the scope of the ultrafast resonance energy transfer in molecular FRET. In the former case the same uorophorevarious biologically relevant environments is the motive ofacts both as energy donor and acceptor, while in the latterthe present studies. case two dierent molecules act as donor and acceptor.In our studies we explored the resonance energy Each donor-acceptor (D-A) pair participating intransfer from one of the uorescent amino acids trypto-FRET is characterized by a distance known as Frster dis- ophan (Trp214) in domain IIA of human serum albumin tance (R0 ) i.e., the D-A separation at which energy trans-(HSA) as a donor to acceptors like photoporphyrin IX fer is 50% ecient. The R0 value ranges typically from 20(PPIX) [11], molecular magnet [12] and quantum dotsto 60 . The rate of resonance energy transfer (kT ) from A(QDs) [13] in FRET based systems. For the enzyme -donor to an acceptor is given by,chymotrypsin (CHT), the choice of the tryptophan as 1 R0 6donor is ruled out as there are seven of them present inkT = (1) D rCHT. The choice of probe like ANS (1-anilinonaphthalene-8-sulfonate) or NPA (4-nitrophenyl anthranilate) was where D is the lifetime of the donor in the absence ofmade on the basis of the selection of a specic molecularacceptor and r is the donor to acceptor (D-A) in case of CHT. NPA is known to bind at the catalytic The rate of transfer of donor energy depends upon thecenter of CHT whereas ANS binds diagonally opposite in extent of overlap of the emission spectrum of the donorposition to the catalytic center. For the investigation of with the absorption spectrum of the acceptor (J()), theFRET in DNA, natural DNA bases are not good choice quantum yield of the donor (QD ), the relative orientationbecause of their very low emission quantum yield. Here weof the donor and acceptor transition dipoles (2 ) and thewill also discuss the labeling of the DNA with uorescentdistance between the donor and acceptor molecules (r)species to make a complete FRET based system and estab-(Fig. 1b). In order to estimate FRET eciency of thelish the fact that the relative orientation of the transitiondonor and hence to determine distances between donor-dipoles of donor (D) and acceptor (A) plays an impor-acceptor pairs, the methodology described below is fol-tant role in the energy transfer when D and A are simul- lowed [16,17]. R0 is given by,taneou