Lecture 6:Fluorescence: Quenching and Lifetimes

Bioc 5085March 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)

Energy changes for absorption are larger than those of emission (due to vibrational relaxation in the excited state)

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)

StokesShift

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 samplehit by the incident light; emitted light (fluorescence) from these molecules may notreach the detection system (or reach it as efficiently). Practical significance of thisis that concentrated samples should be avoided (typical samples concentrations 1 M 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 aminoacids, only trp, tyr, and phe have non-zero fluorescencequantum yields)

For compounds that are fluorescent, they vary greatly in their quantum yields (e.g. f,trp = 1.4f,tyr = 5f,phe)

“Quenching” is a general term that refers to the diminishment or loss offluorescence due to non-radiative processes.

-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: 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 Stern-Volmer equation:

F0/F = 1 + KSV[Q]

where F0 and F are the fluorescence intensities observed in the absence and presence, respectively, of quencher, [Q] is the quencher concentration and KSV is the Stern-Volmer quenching constant.

Most collisional quenchers function by orienting around and stabilizing theexcited state dipole, extending its lifetime, and thus promoting vibrational relaxation (exception to this is oxygen, which functions by promotingthe 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: Collisional or Dynamical Quenching

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).

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, 10328-10338 (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)].

Changes in f as a probe for structural changes