1. Molecular Spectroscopy: Principles and Biophysical Applications BiCh132 Fall Quarter 2012 Jack Beauchamp Many of the illustrations and tables used in these presentations were taken from the scientific literature and various WWW sites; the authors are collectively acknowledged. This presentation is adapted in part from BiCh132 lectures of Professor Barton. Recommended text: “Principles of Fluorescence Spectroscopy” by J. R. Lakowicz (3 rd Edition; 2006) Molecular Probes Handbook -11th Edition (Invitrogen)

2. Introduction to Fluorescence Spectroscopy Useful probe of: environment structure dynamics chemical reactions Timescales: visible absorption~ 10 -15 sec vibrations ~ 10 -14 sec emission~ 10 -9 sec for allowed transitions 10 -6 -10 -3 sec for forbidden transitions On these timescales, emission is sensitive to competing processes

3. 32103210 32103210 Absorption 10 -15 s Fluorescence 10 -9 s Solvent Collisional vibrational dissipation ~ 10 -12 s S1S1 T1T1 Phosphorescence 10 -6 – 10 -3 s S0S0 Intersystem crossing Simplified Energy Level Diagram (Jablonski Diagram)

4. Franck–Condon principle energy diagram. Since electronic transitions are very fast compared with nuclear motions, vibrational levels are favored when they correspond to a minimal change in the nuclear coordinates. The potential wells are shown favoring transitions between v = 0 and v = 2.potential wells Franck-Condon Principle for Electronic Transitions

5. Schematic representation of the absorption and fluorescence spectra corresponding to the energy diagram in previous slide. The symmetry is due to the equal shape of the ground and excited state potential wells. The narrow lines can usually only be observed in the spectra of dilute gases. The darker curves represent the inhomogeneous broadening of the same transitions as occurs in liquids and solids. Electronic transitions between the lowest vibrational levels of the electronic states (the 0-0 transition) have the same energy in both absorption and fluorescence. Franck-Condon Principle for Electronic Transitions

6. Edward Condon Franck-Condon Principle for Electronic Transitions (1926) Classically, the Franck–Condon principle is the approximation that an electronic transition is most likely to occur without changes in the positions of the nuclei in the molecular entity and its environment. The resulting state is called a Franck–Condon state, and the transition involved, a vertical transition. The quantum mechanical formulation of this principle is that the intensity of a vibronic transition is proportional to the square of the overlap integral between the vibrational wavefunctions of the two states that are involved in the transition.

7. Edward Condon

8.  F =  fluorescence quantum yield = fraction of singlets relaxing from excited state via fluorescence # photons emitted by fluorescence unless some catalytic chemiluminescent process Fluorescence Intensity x # excited state molecules  x  c I 0 k F = rate of spontaneous emission P 00 = transition probability same path for excitation and emission # photons absorbed = = Rate constant for emission k F +  (rate constants for non-radiative pathways) Fluorescence Quantum Yields

9. 1. Internal conversion, k ic collision with solvent dissipation of energy through internal vibrational modes basically transfer into excited vibrational states of S 0 Note - k ic increases with T therefore  F decreases with T What processes compete with fluorescence? kFkF k ic kqkq k is S0S0 S1S1 T1T1

10. kFkF k ic kqkq k is S0S0 S1S1 T1T1 2. Intersystem crossing, k is spin exchange converts S to T get slow spin-forbidden phosphorescence for metal complexes often a mixture of states so “luminescence” 3. Collision with quencher, k q e.q. S 1 +Q S 0 +Q* molecules can quench excited state by: energy transfer spin exchange (paramagnetic + spin orbit coupling) electron transfer or proton transfer (+ energy)

11. Note - k F is not temperature dependent but all else is So, what matters are the rates of these competing processes

12. Also, Decay Kinetics of S 1 Suppose initially have concentration in S 1 of S 1 (0) then turn off light Integrating, where If no other processes except fluorescence, fluorescence lifetime (measurable) then Radiative lifetime

13. Can measure steady state or time resolved emission For lifetimes:- then flash and turn off light and measure decay as a function of time - flash photolysis - single photon counting - streak cameras - time resolution depends on flash (also frequency domain measurements - phase modulation) For quantum yields, need geometry constant and correct for emission detectors -use standards (actinometry) Practical things: Excitation Monochromator Emission Monochromator Detector Light source Emitted light Sample

14. Principle: When a fluorescent molecule is excited with plane polarized light, light is emitted in the same polarized plane, provided that the molecule remains stationary throughout the excited state (which has a duration of 4 nanoseconds for fluorescein). If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. If vertically polarized light is exciting the fluorophore, the intensity of the emitted light can be monitored in vertical and horizontal planes (degree of movement of emission intensity from vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule). If a molecule is very large, little movement occurs during excitation and the emitted light remains highly polarized. If a molecule is small, rotation and tumbling is faster and the emitted light is depolarized relative to the excitation plane. Fluorescence Polarization / Depolarization Practical (sometimes annoying) things:

15. Schematic representation of FP detection. Monochromatic light passes through a vertical polarizing filter and excites fluorescent molecules in the sample tube. Only those molecules that are oriented properly in the vertically polarized plane absorb light, become excited, and subsequently emit light. The emitted light is measured in both the horizontal and vertical planes. Fluorescence Polarization / Depolarization

16. Here I ll is the intensity of emitted light polarized parallel to the excitation light, and I ⊥ is the intensity of emitted light polarized perpendicular to the excitation light. An important property of the polarization that emerges from this equation is that it is independent of the fluorophore concentration. Although this equation assumes that the instrument has equal sensitivity for light in both the perpendicular and parallel orientations, in practice this is not the case. Fluorescence Polarization / Depolarization

17. Sarah A. Weinreis, Jamie P. Ellis, and Silvia Cavagnero, Dynamic Fluorescence Depolarization: A Powerful Tool to Explore Protein Folding on the Ribosome, Methods. 2010, 52(1): 57–73. doi: 10.1016/j.ymeth.2010.06. 001

18. Schematic depiction of a protein folding reaction in the cytoplasm of an E. coli cell, showing vividly how different the environment is from dilute in vitro refolding experiments. The cytoplasmic components are present at their known concentrations. Features of particular importance to the folding of a protein of interest (in orange) are: the striking extent of volume exclusion due to macromolecular crowding, the presence of molecular chaperones that interact with nascent and incompletely folded proteins (GroEL in green, DnaK in red, and trigger factor in yellow), and the possibility of co-translational folding upon emergence of the polypeptide chain from the ribosome (ribosomal proteins are purple; all RNA is salmon). The cytoplasm image is courtesy of A. Elcock. Anne Gershenson and Lila M. Gierasch, Protein Folding in the Cell: Challenges and Progress, Curr Opin Struct Biol. 2011, 21(1):32–41. http://dx.doi.org/10.1016/j.sbi.2010.11.001

19. Practical things (for a few $ more): http://www.olympusfluoview.com/applications/fretintro.html

20. Excitation Spectrum – the excitation wavelength is scanned while the emission wavelength is held constant Emission Spectrum - the emission wavelength is scanned while the excitation wavelength is held constant - often gives the mirror image of the absorption spectrum Mirror generally holds because of similarity of the molecular structure and vibrational levels of S 0 and S 1 Given the Franck-Condon Principle, electronic transitions are vertical, that is they occur without change in nuclear positions. If a particular transition probability between 0 and 2 vibrational levels is highest in absorption, it will also be most probable in emission. Stokes shift: fluorescent emission is red-shifted relative to absorption

21. Some Exceptions to Mirror Image Rule 1. Contaminants !! 2. Excitation to higher state(s) S 2 3. Different geometry in excited state 4. Exciplexes (CT state) 5. Excimers 6. pK effects (excited state acid base properties) Dimer excited state

22. Acid-base properties are modified in electronically excited states Example- pKa for acridine in ground state= 5.5 pKa for acridine in excited state= 10.7 protonation can occur during excited state lifetime Effects are quantified with use of the Förster Cycle Think of some applications of this phenomenon

23. Förster Cycle: Quantifies changes in acid-base properties in electronically excited states ArOH (aryl alcohol such as napthol) – The shift in absorption spectra of the acid and its conjugate base can be used to quantify the difference in pKa in the ground and excited electronic state

24. . Fluorescent Probes Absorption and emission spectra of biomolecules. Top: Tryptophan emission from proteins. Middle: Spectra of extrinsic membrane probes. Bottom: Spectra of the naturally occurring fluorescence base, Yt base. DNA itself(---) displays very weak emission

25. Normally use extrinsic probes or modified bases/ unnatural amino acids (check out the Molecular Probes Catalogue) when intercalated, yield and lifetime increase Probe Dansyl chloride Ethidium max  max (x10 -3 ) max FF  F (ns) AbsorptionFluorescence 340-350 274 4.3 1.4 510-560 303 0.1-0.3 0.05 10-15 2 + DNA ~1 20

26. If you have 2 fluorescent components (probes), even two bound components, they will have different rates of quenching, k q k q gives measure of accessibility of chromophore F1 F2 Q k q for F1 > k q for F2 Fluorescence Quenching

27. In the absence of quencher, in the presence of quencher, where quenching is the result of bimolecular collisions. Stern-Volmer Analysis of Quenching

28. Stern-Volmer quenching with concentration of Q, [Q] Stern-Volmer Plot [Q][Q] 1 Slope= K SV where K SV = k q  

29. expect k q ’s of 10 10 M -1 s -1 or less Values of k q reflect collisional frequency and bimolecular diffusion controlled rate constant, k 0 Smoluchowski eqn. R = collisional radii D = diffusion coefficients k q = f Q k 0 f Q = quenching efficiency if f Q = 0.5, 50% of collisions lead to quenching Can estimate D from Stokes-Einstein eqn.

30. Q + F FQ Consider equilibrium formation of a ground state complex which is not fluorescent: The total conc. of fluorophore = or If FQ is not fluorescent, thenfraction of fluorescence

31. so that gives same K S.V. as But could have or even [Q][Q] [Q][Q] 1 Slope= K S.V. [Q][Q][Q][Q]

32. For dynamic quenching, quenching process is diffusion controlled For static quenching but no change in  – not a diffusion controlled process [Q][Q][Q][Q] DynamicStatic

33. Singlet-Singlet Energy Transfer (Förster Transfer)

34. Singlet-Singlet Energy Transfer (Förster Transfer)

35. Singlet-Singlet Energy Transfer (Förster Transfer) Very useful for “long range” distance (20-80 Å) R R Donor Acceptor

36. D* D0D0 A* A0A0 Pick donor and acceptor to have appropriately matched energy levels: DD A A Absorption Emission k T = rate constant for transfer D* +A 0 D 0 +A* kTkT k -T k -T is not likely given rapid vibrational relaxation Energy transfer gives sensitized emission and donor deexcitation Resonant interaction with acceptor excitation- weak coupling limit

37. Real world example: Cyan fluorescent protein/Red fluorescent protein Absorption and emission spectra of cyan fluorescent protein (CFP, the donor) and red fluorescent protein (RFP or DsRed, the acceptor). Whenever the spectral overlap of the molecules is too great, the donor emission will be detected in the acceptor emission channel. The result is a high background signal that must be extracted from the weak acceptor fluorescence emission.

38. What’s the basis for the interaction? -As in exciton coupling, dipole-dipole: just weak coupling limit Can describe the potential operator Where R is distance between A + D and are dipole moment operators lump all geometric and orientational parameters in here- really hard to know , lots of variability  = 0-4

39. According to Fermi’s Golden Rule: -rate of transition is proportional to the square of the expectation value for the interaction causing the excitation. emission absorption for isoenergetic D(b a) emission A(a b) excitation quantum yield lifetime of donor w/o acceptor frequency of transition extinction coefficient for A

40. For general case, where transition involves a range of frequencies where and or normalized fluorescence of donor overlapping with acceptor refractive index of medium between donor and acceptor

41. Naively, looks like D is emitting and A is reabsorbing but that transfer is trivial. Also what would be effect on ? Usual to define efficiency

42. get 1/R 6 dependence for E can measure 10-100 Å distance separations depending on FRET pair Want to measure donor-acceptor partners near R 0 depending on experiment This yields largest change in E for small changes in R that occur in the given experiment.

43. Very unique distance regime - FRET provides a spectroscopic ruler

45. Selected Applications of FRET Structure and conformation of proteins Spatial distribution and assembly of protein complexes Receptor/ligand interactions Immunoassays Probing interactions of single molecules Structure and conformation of nucleic acids Real-time PCR assays and SNP detection Detection of nucleic acid hybridization Primer-extension assays for detecting mutations Automated DNA sequencing Distribution and transport of lipids Membrane fusion assays Membrane potential sensing Fluorogenic protease substrates Indicators for cyclic AMP and zinc - Molecular Probes website

46. Different ways to carry out experiment: monitor quenching of donor and/or enhanced emission by acceptor 1.) Quenching of donor D alone D+A E= fraction of donors deexcited therefore 1-E= fraction of donors remaining excited 2.) Enhanced emission by acceptor -should be sensitized emission: excite D, watch A emit D absorb Acceptor emission watch here I I

47. D alone D+A A alone donor quench A sensitized emission An example: Distance measurement in melittin Depending upon solvent, can exist as monomer or tetramer,  -helix or random coil In practice, want 3 replicas for study:

48. Overlap integral (shaded area) for energy transfer from a tryptophan donor to a dansyl acceptor on melittin. R 0 =23.6 Å Determine overlap integral for trp/dansyl pair: R 0 = 23.6 Å

49. E=0.45 R=24.4Å

50. But there are issues- 1.)  2 is not known, nor directly measurable for so even rough estimate suffices Dale Eisinger Method- exploit the jitter Likely there is fast geometric averaging before transfer, blurring  2 often set  2 =2/3 for dynamic avg. of all geometries means uncertainty in R is < 15% macromolecule acceptor donor κ is related to the relative orientation of the donor/acceptor pair

51. 2.) Imperfect Stoichiometry 3.) Does the probe perturb the structure? if possible it is good to rely on intrinsic probes: in a protein tyr/trp energy transfer is possible D A1A1 A2A2 k T1 k T2 (monomer/ tetramer equilibrium for example)

52. Classic papers: A Spectroscopic Ruler Lubert Stryer and Richard Haugland Proc. Natl. Acad. Sci USA, 58, 719-726 (1967)

53. D A

54. A = magnitude of excitation =  a + E x  d Without the donor, excitation is that of acceptor; in the presence of donor, see sensitized emission and therefore excitation includes that of donor.

56. Mapping the Cytochrome c Folding Landscape Julia G. Lyubovitsky, Harry B. Gray,* and Jay R. Winkler* JACS, 2002, 124, 5481 Measurements of FRET between heme and C-terminal dansyl

58. There is a rapid equilibration among extended conformations, enabling escape from frustrated compact structures Some population of extended conformations, with long distances remain even at long times.

59. Single Molecule Fluorescence Experiments

60. Example: Nucleic Aid Conformation and dynamics Single molecule FRET study of Holliday junction by total internal reflectance microscopy. The nucleic acid is tethered to the surface via biotin-neutravidin conjugation. The conformational dynamics is shown in the fluorescence time trace. McKinney, Declais, Lilley, Ha, Nature Structural Biol. 10 93-97 (2003) TIRF Area detector/ camera