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Emission Spectroscopy and Molecular Motions in Biomolecular Systems. W.C. Galley, Dept. of Chemistry. Base. Acid. Base. Neutral. Neutral. Acid. EXCITATION WAVELENGTH (nm). Fluorescence Spectrum of ANS in Differing Local Environments. F. x30. 450 500 550.

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slide1

Emission Spectroscopy and Molecular Motions in Biomolecular Systems.

W.C. Galley, Dept. of Chemistry

slide3

Base

Acid

Base

Neutral

Neutral

Acid

slide10

Fluorescence Spectrum of ANS in Differing Local Environments.

F

x30

450 500 550

wave.length (nm)

slide12

Fluorescence Spectrum of ANS in Differing Local Environments.

F

glycerol/H2O at 296K

x30

450 500 550

wave.length (nm)

slide13

Fluorescence Spectrum of ANS in Differing Local Environments.

glycerol-H2O at 77K (rigid glass)

F

glycerol/H2O at 296K

x30

450 500 550

wave.length (nm)

slide16

The fluoresence of pyrene displaying the presence of both the monomer fluorescence as well as an excimer band. The latter arises from the formation of a complex between a molecule in the excited singlet state and a monomer.

slide18

The contribution of the “excimer” to the monomer band in the fluorescence of pyrene in e.g. a membrane is dependent on the ability of the probe to diffuse during the long pyrene excited singlet-state lifetime of ~ 200 nsec. (t for Trp ~ 4 nsec)

slide19

Pyrene forms excited- and ground-state dimers (excimers). The excimer displays its own red-shifted fluorescence.

  • b) Pyrene has an exceeding long excited-singlet state (“fluorescence”) lifetime. It has the opportunity to diffuse and encounter partners.
  • c) The data provide a measure of viscosity of the media e.g. lipid bilayer.
slide20

The Measurement of Forster (FRET) Transfer Efficiencies from Donor Quenching.

In the presence of a single donor and acceptor:

fluorescence intensity of the donor in the presence of acceptor.

fluorescence intensity of the donor alone.

slide22

Fdonor

Fdonor + coenzyme

slide26

Decomposition of the Trp decay in the presence of coenzyme into a bi-exponential decay

Time (nsec)

slide28

chromophores with their transition dipoles randomly distributed

chromophores photo-selected with transition dipoles in the vertical direction.

F║

F┴

F┴ >

F║

slide29

Steady-state emission anisotropy A can be represented in the form of the Perrin Equation:

where Ao = the anisotropy anticipated for a rigid particle.

= 0.4 when the absorption and emission transition . dipoles are in the same direction (parallel).

= - 0.2 when the transition dipoles are perpendicular

and tF = the lifetime of the excited state.

Note: when A = 0.5Ao, f = tF

slide30

f = the rotational correlation time. It is a measure of the time t it takes for the molecule to forget where it was, in a rotational sense, at t = 0.

From an Equation due to Einstein f for a spherical particle is given by:

V is the volume of the particle, h = the viscosity of the solvent in which it is swimming.

For a spherical particle A(t) decays exponentially:

slide31

Inverting the equation results in a form that is linear in 1/f:

or

So that a plot of 1/A versus T/hresults in a linear equation .if there is a single rotational correlation time. The plot is generated by measuring A while varying h and/or T. The solvent viscosity can be varied by adding sucrose or e.g. glycerol to a buffer. tF must be known and Ao can be obtained from the intercept.

slide32

Decay of the vertically- and horizontally-polarized components of the fluorescence of a labelled globular protein.

vertically-polarized excitation

slide34

The exponential decay indicates a single rotational correlation time fcharacteristic of isotropic rotations. With a rigidly-bound label the rotational correlation time reflects the overal rotation of the globular protein as a hydrated sphere:.

A non-sperical object displays a non-exponental decay that might contain as many as 5 components, with a rod-like particle possessing 3 rotational correlation times.