Förster Energy Transfer - AKA - Fluorescence Resonance Energy Transfer

Transcription

1 örster Energy Transfer - K - luorescence esonance Energy Transfer 1. Origins: Theory of Energy Transfer developed by T. örster (örster nnalen der Physi. :55-75.). evelopment of ET as a Spectroscopic uler. (Stryer & Haugland Proc. Natl. cad. Sci. 58: ) 3. Some ecent pplications: a. luorogenic Energy Transfer Substrate (Hasegawa et al Proc. Natl. cad. Sci US 100: ) b. ET & Single Molecule ibozyme ction (Zhaung et al. 00. Science 96: ) c. Single Molecule Protein olding (Lipman et al Science 301: )

2 Energy Transfer Efficiency depends on Separation istance (r -6 ). QM dipole-dipole operator has an r -3 dependence, but the probability of transfer is proportional to the square of the expectation value; thus a r -6 dependence.. Orientation. ipole-dipole interactions are orientation dependent. 3. Spectral Overlap. The emission band of the donor must overlap with the absorption band of the acceptor. 4. Quantum Yield. luorescence yields of donor and acceptor should be high for efficient transfer. 5. Non-overlapping bsorption Bands, Non-overlapping emission bands. Not a necessity, but simplifies life. T x 10 ( Jκ n ) cm 0 φ 1/ 6 where lifetime of the donor in the absence of acceptor J is the overlap integral κ is the orientation factor à /3 rapid tumbling limit φ is quantum yield of the donor in the absence of acceptor

3 onor bsorption Band onor Emission Band cceptor bsorption Band cceptor Emission Band λ

4 elaxation following à transfer lowers the probability of reverse transfer T b + a -T a + b 1. bsorption (10-15 s) b.. Vibronic elaxation (10-1 s) 3. Energy Transfer (One Coupled Transition) (10-15 s) b Vibronic elaxation (10-1 s) 5. dditional Coupled Transitions a a

5 Isolated chromophore lifetime & quantum yield φ + ic + is + q [ Q] luorescence Quantum Yield ba 1 The intrinsic fluorescence rate constant is inversely proportional to the time constant for radiative decay, φ where + ic 1 + is + q [ Q] The observed fluorescence time constant,, is the inverse of the observed rate constant. Two chromophores efficiency of transfer E T + + T ic + is

6 Measurement of transfer efficiency (1) φ+ + ic + is 1 φ ic is T E elative quantum yields of donor (φ ) and donor in the presence of acceptor (φ + ) φ φ + λ

7 Measurement of transfer efficiency () onor/cceptor excitation scan in the presence of the acceptor is affected by the presence of the donor ε C φ + ε C λ Eφ ( + ) ( ) 1 + ( ε C ε C )E Measurement of transfer efficiency (3) ( + ) 1 E luorescence lifetime of donor is shortened in the presence of acceptor

8 What is T? ) T ~ ( a b b a V ν T is proportional to the square of the expectation value for the interaction causing the excitation Where ( ) ( )( ) 5 3 ˆ ˆ ˆ ˆ ˆ ~ V 3 ˆ ˆ ~ V κ ) 3 T ˆ ˆ ( a b b a κ ν ) 6 T ˆ ˆ ( a b b a κ ν The orientation and distance contributions to the dipole-dipole operator are separated, removed from the integral, which are then also factored

9 onor & cceptor Contributions to the Overlap Integral ab 3 ε ˆ b a 9.18 x 10 dν ν ε ν b a cceptor The square of the expectation value for the acceptor is ipole Strength of the acceptor.. at a single frequency onor The donor undergoes spontaneous emission (fluorescence). The Einstein coefficient for spontaneous emission is related to the ipole Strength π ν c 3 ba 3 ab φ + ic + is + q ba ba ab 3 ab 3 since ν ν [ Q] a ˆ b φ à 3 ν 1 φ 1 ba T ( T ( κ ν 6 φε ν ) 4 κ φ ε ν ) f 6 4 ν band κ φ 6 ( v) dν J Together These contributions (integrated over the acceptor absorbance band) produce the overlap integral (J) the orientation factor (κ ) and the dependence of the donor quantum yield and lifetime (φ, ), as well as the -6 distance dependence.

10 . evelopment of ET as a Spectroscopic uler. (Stryer & Haugland Proc. Natl. cad. Sci. 58: ) Well-defined donor acceptor pair in a specifically labeled homologous series of Polyproline II helix. (ssume that 0 is constant.) Merrifield Solid Phase Synthesis of Peptide Bruce Merrifield developed the solid phase method, but who pioneered the tboc (and now moc) protecting groups used in peptide synthesis? nswer: Lou Carpino (UMass Chem ept.)

11 bsorption and Emission Spectra of onor and cceptor

12 Excitation Scans measure Transfer Efficiency ( + ) 1 + (ε C ε C )E ( ) ( + ) ε E 1 ( ) ε

13 E has a 1/6 dependence 06 E E 0 1 ln 1 n ln( ) ln 0n E ( )

14 Single Molecule ET (n Example, Zhaung et al.)

15 In Vivo ET (n Example, Hasegawa et al.)