Fluorescence spectroscopy. Introductory lesson

1
Fluorescence spectroscopy.Introductory lesson

• Lecture 11
• October 6th

2
Announcement(s)
1) On Oct 20th Ill be away from the city. This
session will be substituted by the seminar from
Chemistry dept. The students will attend the
seminar that will be held by the theoretical
chemist Harold Scheraga (Cornell), in Chemistry
on October 11th, 400 PM, Room 1-019, Center for
Science Technology
The term paper submission will be accompanied by
a short comprehensive talk (20 min 5 min qns)
that represents an overview in the field. This
will be a good practice for other future
occasions. Two future sessions will be devoted
to your talks!
3
Single-molecule approaches used (so far) to study
chromatin structure and dynamics (Zlatanova and
Leuba)
4
Concept of FRET, single-dye fluorescence and
magnetic tweezers
concept of FRET
a
c
b
external magnets
evanescent field
N
S
intensity
magnetic bead
DNA
spectra of donor and acceptor dyes and energy
transfer
donor dye absorbance emission
acceptor dye absorbance emission
surface
objective
N
S
normalized absorbance
evanescent field
wavelength of light
spFRET data for reversible interactions
fluorescence intensity
objective
time
5
Fluorescence widely used to study proteins

• Advantages
• Fluorescence signals are very sensitive to the
  environment of the probe
• Requires little sample
• Time scale is in the nanosecond range
• Easy to use
• Disadvantages
• Does not give detailed structural information

6
What can light tell us about a molecule and its
environment?
The basic concept is that after a molecule
absorbs light, it takes time for it to re-emit
light. During this time, various things can
happen to the molecule. These things include
1) solvent rearrangement 2) rotation of the
molecule) 3) loss of energy to neighboring
molecule (energy transfer) 4) reaction
(quenching)
7
Electrons change orbitals
light
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Important points

• Absorption of light is fast 10-15 sec
• Light absorption at the lowest energy causes
  electrons to go from the highest occupied
  molecular orbital to the lowest unoccupied
  molecular orbital.
• Light absorbs at a given orientation. This means
  that differently polarized light are
  preferentially absorbed

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Energy diagram
S2
Electronic levels
S1
Electronic vibrational -gt vibronic
Absorption
Fluorescence
l
10-9 sec
l
10-15 sec
Internal conversion
l
Vibrational levels
So
10
fluorometer
monochromator
lamp
sample
Fluorescence usually measured at right angles
photomultiplier
monochromator
Fluorometers usually have two monochromators
One for excitation and one for emission
Intensity
excitation
emission
Wavelength, nm
11
Time-resolvedfluorometer
sample

laser
Intensity
electronics
photomultiplier
monochromator
t lifetime
Exponential decay I Ioe-t/t
Time (nanoseconds)
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Natural fluorescent chromophores
Amino acids
Cofactors
tryptophan
tyrosine
phenylalanine
flavin
13
Tryptophan fluorescence indicates location
Typical result
Nuclease
Folded protein
Unfolded protein
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W99F
Trp Repressor
W19F

• Unfolding Trp exposed to water
• Trp at position 19 in more stable part of the
  protein than Trp at position 99

15
Reference
Joseph R. Lakowicz, Principles of Fluorescence
Spectroscopy, Second Edition, Kluwer
Academic/Plenum Publishers, New York, 1999.