Luminescence methods Methods of biophysical chemistry - seminar Jan Novotný novotnyjan@mail.muni.cz November 13, 2019 J. Novotný November 13, 2019 1/11 Energetic digram Fill in attached diagram and compare the phenomena according to parameters indicated in a table: Phenomenon time regime k vs. kvit, order A Absorption Fluorescence Phosphorescence J. Novotný November 13, 2019 2 / 11 Energetic digram Fill in attached diagram and compare the phenomena according to parameters indicated in a table: $2 Si Absorption I Internal i Conversion j_ Intersystem Irossing Fluorescence Phosphorescence. Phenomenon time regime k vs. kvib order Xmax Absorption 10-15s > 1 Fluorescence 10-9s < 2 Phosphorescence 10°s < 3 J. Novotný November 13, 2019 2 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. O Rotational correlation time is lower for bulky molecules in viscous environment. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. O Rotational correlation time is lower for bulky molecules in viscous environment. O Stokes shift arises from non-radiative vibrational relaxation. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. O Rotational correlation time is lower for bulky molecules in viscous environment. O Stokes shift arises from non-radiative vibrational relaxation. 0 The fluorescence spectrum does not depend on the wavelength of excitation radiation. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. O Rotational correlation time is lower for bulky molecules in viscous environment. O Stokes shift arises from non-radiative vibrational relaxation. 0 The fluorescence spectrum does not depend on the wavelength of excitation radiation. O Resonance transfer of energy (RET, FRET) occurs through the reabsorbtion of photon between donor and acceptor. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. O Rotational correlation time is lower for bulky molecules in viscous environment. O Stokes shift arises from non-radiative vibrational relaxation. 0 The fluorescence spectrum does not depend on the wavelength of excitation radiation. O Resonance transfer of energy (RET, FRET) occurs through the reabsorbtion of photon between donor and acceptor. O Fluorescence life time is inversely proportional to sum of rate constants of all active radiative and non-radiative pathways. J. Novotný November 13, 2019 3 / 11 Starting problems: Are the following statements true or false. Explain your decision. O Molecules characterized by active fluorescence are usually rigid aromatic compounds of limited vibrational freedom. Q Phosphorescence transition is defined as emission of photon from singlet state. O Rotational correlation time is lower for bulky molecules in viscous environment. O Stokes shift arises from non-radiative vibrational relaxation. 0 The fluorescence spectrum does not depend on the wavelength of excitation radiation. O Resonance transfer of energy (RET, FRET) occurs through the reabsorbtion of photon between donor and acceptor. O Fluorescence life time is inversely proportional to sum of rate constants of all active radiative and non-radiative pathways. O The measurement of fluorescence anisotropy employs circularly polarized excitation radiation J. Novotný November 13, 2019 3 / 11 Exercise 1 Assign displayed fluorescence probes to corresponding abbreviation and biochemical application DAPI, ddATP-Dye, DPH, TNS Exercise 1 Assign displayed fluorescence probes to corresponding abbreviation and biochemical application TNS ddATP-dye probing proteins nucleic acid sequencing DAPI, ddATP-Dye, DPH, TNS Exercise 2: Fluorescence methods Assign the appropriate methods exploiting fluorescence techniques to following tasks: A) Determination of hydrodynamic radius of a protein. B) DNA hybridization. C) Localisation of Trp residue (on surface or inside a protein). D) Portion of unsaturated phospholipids in biomembrane. E) Determination of Ka of eosin dimerisation. Correlation time of fluorescence label, fluorescence anisotropy of DPH, Stokes shift, emission of excimer, FRET J. Novotný November 13, 2019 5 / 11 Exercise 2: Fluorescence methods Assign the appropriate methods exploiting fluorescence techniques to following tasks: A) Determination of hydrodynamic radius of a protein. Correlation time of fluorescence label B) DNA hybridization. FRET C) Localisation of Trp residue (on surface or inside a protein), fluorescence decay, Stokes shift D) Portion of unsaturated phospholipids in biomembrane. viscosity-anisotropy of DPH E) Determination of of eosin dimerisation. emission of excimer Correlation time of fluorescence label, fluorescence anisotropy of DPH, Stokes shift, emission of excimer, FRET J. Novotný November 13, 2019 5 / 11 Exercise 3: FRE" Try to interpret following fluorescence experiment carried out on complex of proteinkinase consisting of catalytic (C) and regulative (R) subunit. Both parts are labelled with probes: unit C with fluorescein (Fl) and unit R with rhodamine (Rh). In native form of R2C2 the FRET can be detected. Identify the direction of transition and explain the effect of cAMP and PKI inhibitor added to the studied sample. J. Novotný November 13, 2019 6 / 11 Exercise 3: FRE" Try to interpret following fluorescence experiment carried out on complex of proteinkinase consisting of catalytic (C) and regulative (R) subunit. Both parts are labelled with probes: unit C with fluorescein (Fl) and unit R with rhodamine (Rh). In native form of R2C2 the FRET can be detected. Identify the direction of transition and explain the effect of cAMP and PKI inhibitor added to the studied sample. J. Novotný November 13, 2019 6 / 11 Exercise 4: Kinetic parameters of fluorescence Eosin fluorophor is characterized by quantum yield 0.65 and fluorescence life time of 3.1 ns. Calculate the life time of radiative, non-radiative transition and intrinsic life time of fluorescence. J. Novotný November 13, 2019 7 / 11 Exercise 4: Kinetic parameters of fluorescence Eosin fluorophor is characterized by quantum yield 0.65 and fluorescence life time of 3.1 ns. Calculate the life time of radiative, non-radiative transition and intrinsic life time of fluorescence. Solution v-\-knr ' v-\-knr Rate constants: f = M5 = 0.21 ns-1 T km T 3.1 -0.21 = 0.11 ns -l J. Novotný November 13, 2019 7 / 11 Exercise 5: Perrin equation - depolarisation of emitted signal Based on assumption of exponential decay of intensity l(t) and anisotropy r(t) of fluorescnce signal derive relation between anisotropy r, life time r and correlation time 6. Use the definition of time-weighted average of anisotropy r as a starting point: J~ r(t)l(t)dt r ~ /~ /(t)dt J. Novotný November 13, 2019 8 / 11 Exercise 5: Perrin equation - depolarisation of emitted signal Based on assumption of exponential decay of intensity l(t) and anisotropy r(t) of fluorescnce signal derive relation between anisotropy r, life time r and correlation time 6. Use the definition of time-weighted average of anisotropy r as a starting point: r = J0°° r(t)l(t)dt Jo°° Kt)dt / = /0e r = r0e e r = /oro/-e ť(' + g)dt _ ro(^ + j) 1 /o/0°°e ídt J. Novotný November 13, 2019 8 / 11 Exercise 6: FRE" The protein human serum albumin (HSA) has a single tryptophan residue at position 214. HSA was labelled with an anthraniloyl group placed covalently on cysteine-34. Emission spectra of the labelled and unlabelled HSA are shown in attached figure. The Förster distance for Trp to anthraniloyl transfer is 30.3Ä. Use the emission spectra in the attached figure to calculate the Trp to anthraniloyl distance. The rate constant of RET can be estimated using formula: kRET = l~ (^)6. 260 340 420 WAVELENGTH ( nm ) J. Novotný November 13, 2019 9 / 11 Exercise 6: FRE" The protein human serum albumin (HSA) has a single tryptophan residue at position 214. HSA was labelled with an anthraniloyl group placed covalently on cysteine-34. Emission spectra of the labelled and unlabelled HSA are shown in attached figure. The Förster distance for Trp to anthraniloyl transfer is 30.3Ä. Use the emission spectra in the attached figure to calculate the Trp to anthraniloyl distance. The rate constant of RET can be estimated using formula: kRET = l~ (^)6. HSA---' » Anthranilate ; 1 \ I 260 340 420 WAVELENGTH ( nm ) Řešení 0 = Kn6 + i Quantum yield 0 at A 340 nm: emission of albumin with acceptor/emission of free form=0.2/0.55 0 = 0.364 = 30.36+r6 r = = 27.6Ä 1.756 J. Novotný November 13, 2019 Exercise 6: Dipolar interaction - orientation dependence Efficiency of resonance transfer depends beside the spectral overlap and spatial distance between donor and acceptor on mutual orientation of transition moments. These moments interact as two dipols: Ha-Hb ~ 3(/M.r)(/ie.r) Calculate the value of orientation factor k2 for attached model. i ,-""1 / 1 J. Novotný November 13, 2019 10 / 11 Exercise 6: Dipolar interaction - orientation dependence Efficiency of resonance transfer depends beside the spectral overlap and spatial distance between donor and acceptor on mutual orientation of transition moments. These moments interact as two dipols: Ha-Hb ~ 3(/M.r)(/ie.r) Calculate the value of orientation factor k2 for attached model. i ,-""1 / 1 Solution k2 = V2 J. Novotný November 13, 2019 10 / 11 Further reading Joseph R. Lakowicz: Principles of Fluorescence Spectroscopy Jihad Rene Albani: Principles and Applications of Fluorescence Spectroscopy P. Atkins, J. de Paula: Physical Chemistry J. Novotný < rS1 ► < -ž ► < -E ► -E -0 0,0 November 13, 2019 11 / 11