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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 = 1.4f,tyr = 5f,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)].