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Lecture 6:
Fluorescence: Quenching and Lifetimes
Bioc 5085
March 28, 2014
Fluorescence Spectroscopy
Flourescence (and a related process called
phosphorescence) occurs when an electron returns to
the electronic ground state from a excited state and loses
its excess energy as a photon.
Jablonski Diagram
Sample Spectrum (Tryptophan)
Stokes
Shift
Energy changes for absorption are
larger than those of emission (due to
vibrational relaxation in the excited
state)
hc
E = hv =
l
v = frequency
c = speed of light
l = wavelength
h = Planck's constant
Hence, absorption occurs at lower
wavelengths than emission; this is
known as the Stokes shift (Stokes
shift is formerly defined as the
difference between the lowest energy
absorbance maximum and the highest
energy emission maximum)
Steady-State Fluorimeter
Inner filter effect: Can occur when the sample is too concentrated … idea is
that all of the available light is absorbed by molecules on the edge of the sample
hit by the incident light; emitted light (fluorescence) from these molecules may not
reach the detection system (or reach it as efficiently). Practical significance of this
is that concentrated samples should be avoided (typical samples concentrations
1 mM or lower).
Fluorescence Quantum Yields
Fluorescence Quantum Yield (f) = photons emitted/photons absorbed
For some compounds, there are no photons emitted; that is, the
fluorescence quantum yield is zero (in the case of the 20 common amino
acids, only trp, tyr, and phe have non-zero fluorescence
quantum yields)
For compounds that are fluorescent, they vary greatly in their
quantum yields (e.g. f,trp = 1.4f,tyr = 5f,phe)
“Quenching” is a general term that refers to the diminishment or loss of
fluorescence due to non-radiative processes.
Quenching: Internal Conversion (Vibrational Relaxation)
-Compounds with a high degree of internal flexibility have very high vibrational
levels of the ground state; this in general promotes internal conversion (accounting
for the lack of any detectable fluorescence by compounds with significant internal
flexibility; also accounting for the lower quantum yields of compounds that have
residual internal flexibility)
-Internal conversion in general increases as the temperature is raised (therefore,
the observed fluorescence will decrease with increasing temperature).
Quenching: Internal Conversion (Vibrational Relaxation)
Quenching: Resonance Energy Transfer
As we will discuss, resonance energy transfer can occur over
distances of up to 50 Å
One of the important consequences of this is that in proteins
that contain both tyr and trp, the emitted fluoresence is
dominanted by that of trp (tyr absorbs and emits at lower
wavelengths than trp, and hence the tyr emission can serve to
excite trp).
Quenching: Collisional or Dynamical Quenching
Collisional or Dynamical Quenching is de-excitation of the excited
singlet state as a consequence of collisions with other groups (collisions
can be intra-molecular or inter-molecular). Collisional quenching is
described by the
where F0 and F are the fluorescence intensities observed in
Stern-Volmer equation: the absence
and presence, respectively, of quencher, [Q] is
F0/F = 1 + KSV[Q]
the quencher concentration and KSV is the Stern-Volmer
quenching constant.
Quenching: Collisional or Dynamical Quenching
Most collisional quenchers function by orienting around and stabilizing the
excited state dipole, extending its lifetime, and thus promoting vibrational
relaxation (exception to this is oxygen, which functions by promoting
the conversion from the excited singlet to triplet states)
Small molecule extrinsic quenchers must in general be particularly effective
to have an effect, since as noted, the lifetimes of the excited singlet states
are typically quite short ( = 1 - 10 ns) compared to the diffusion limit for
small molecules in aqueous solution (5 x 10-5 cm2 s-1).
Common extrinsic quenchers include Cs+, I-, NO3-, and
acrylamide (CH2=CH-CONH2).
Common intrinsic quenchers include internal protonated carboxylate
groups (side chains of Asp or Glu) and side-chain amide groups (Asn
and Gln).
Quenching: Propensity to Phosphoresce
Singlet state: All electrons are spin-paired
Triplet state: One set of electrons is not spin-paired
Note: Lifetimes of excited triplet states are very long ( ~ 10 s) compared to
lifetimes of excited singlet states ( ~ 1 - 10 ns); thus phosphorescence is quite rare
since internal conversion and other quenching processes (see previous few slides)
provide competing non-radiative mechanisms that lead to the release of energy.
Fluorescence as a tool for studying the structure and
dynamics of biological macromolecules
-Fluorescence (in particular, the degree of quenching) is generally much
more sensitive to the environment than is absorption; therefore, it is one of
the most powerful tools for studying ligand binding or conformational
changes.
-Sensitivity is due in large part to the fact that the lifetime ()
of the excited (singlet) state (1 - 10 ns) is often comparable to the
timescale of many processes that occur in proteins, such as
protonation/deprotonation, local conformational dynamics, and overall
rotation and translation (in contrast, absorption occurs on timescales on the
order to 10-15 sec; hence the molecule is essentially fixed in the course of
this spectroscopic measurement).
Changes in f as a probe for structural changes
Fluorescence emission spectra of ethidium bromide in the absence (dotted
lines) or presence of double-stranded DNA (panel a; solid line) or RNA
(panel b; solid line).
Changes in f as a probe for structural changes
Figure 3. Representative guanidinium chloride unfolding curves for the four disulfide mutants of
nuclease in their oxidized and reduced states. Intrinsic fluorescence emission intensities at 325 nm
were recorded, at a temperature of 294.5 K, as a function of increasing concentration of GdmCl. Filled
symbols denote data collected for samples under oxidizing conditions, whereas open symbols
correspond to data collected in the presence of 10 mM DTT. [Hinck, et al., Biochemistry, 35, 1032810338 (1996)].
Changes in f as a probe for structural changes
Figure 1. Bis-ANS fluorescence emission spectra. Fluorescence emission
spectra of (a) bis-ANS (5 M), (b) bis-ANS (5 M) + apical domain (5 M), (c)
bis-ANS (1 M) + apical domain (5 M) + urea (2.5 M), and (d) bis-ANS (5 M)
+ apical domain (5 M) + urea (5.0 M), all in a 10 mM sodium phosphate
buffer, pH 7.0. [Smoot, et al., Biochemistry, 40, 4484-4492 (2001)].