4 fluoresence spectroscopy

Further study;

What are the important differences between the design of a spectrophotometer and a fluorimeter? Which amino acids can be used as intrinsic fluorophores in proteins? Rank them best to worst with regard their fluorescence emission at an identical [fluorophore] Review the differences between radiative decay and non-radiative decay from the first electronic excited state to the ground electronic state using a Morse diagram Review the different processes that can lead to non-radiative decay from the first electronic excited state Review the concept of fluorescence lifetime (t) as this will be important for understanding fluorescence anisotropy in part 2 How do you think increasing the temperature will affect the fluorescence lifetime of a fluorophore?

Theory of fluorescence

- Absorption of a photon - Electron into first excited state - Election energy lost as heat - lose energy by moving through the vibrational levels without needing to radiate any energy - lost by Collison with solvent, rotation within the molecule bonds or bending of bonds. - Fluorescent (radiative); electron can only go from first excited state to ground state by emitting fluorescence (->), lose energy by dropping through vibrational energy levels, as no overlap between the excited and ground undergoes radiative decay to the ground state releasing energy of a longer wavelength/lower energy than that which it absorbed in the first place to get to the excited state. - inNon-fluorescent (Non-radiative); electron (photon) goes from first excited state to ground state without emitting radiation

Florescence anisotropy to probe protein-protein interactions;

- CRP(protein) has fluorophore, tumbling rapidly and is highly depolarised but if you add RNA polymerase, see anisotropy increases as the protein is no longer tumbling quickly due to its bonds to the RNA polymerase (has rotational correlation time consistent with RNA polymerase). - Can be used to measure binding between two species.

Forster Resonance Energy Transfer (FRET)

- Arises when two fluorophores with the appropriate spectral properties are in close proximity - FRET occurs when the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor - FRET is a form of dynamic donor fluorescence quenching - Excite lambda max for to donor fluorophore instead of getting fluorescence emission at a wavelength that's consistent with the donor theres non radiative transfer of energy for the donor fluorophore to the acceptor fluorophore, so only see fluorescence consistent with the acceptor fluorophore. - A donor chromophore is its excited state can transfer energy by a non-radiative, long-range, dipole-dipole coupling mechanism to an acceptor chromophore in close proximity (< 10 nm) - Theodor Förster (May 15, 1910 to May 20, 1974) was a German physical chemist - FRET = Förster resonance energy transfer - Distance dependence is very sensitive so it can be used as a spectroscopic ruler - measure distances between two fluorophore sites in macromolecules or complexes. But need the donor and acceptor fluorophores at specific sites in your species of interest so need good idea of structure. - If the donor is irradiated with light at λex, donor and FRET is possible, then emission occurs not only at λem, donor but also at λem, acceptor

Fluorescence Depolarisation by Rotational Diffusion

- As movement increases depolarisation increases become isotropic quickly with two measured values equivalent faster. - Rotational correlation time (tc) is the time it takes a particle to rotate by one radian (≈ 57.3˚). - As molecular weight of a globular protein increases the rotational correlation time also increases, so it takes more time for it to rotate through one radian. - Average density for proteins (r) = 1.37 g/ml - h = solution viscosity - So fluorescence measurements are exclusively made with extrinsic dyes that have long fluorescence lifetimes.

Extrinsic fluorescent probes;

- Desirable properties of an extrinsic fluorophore; o Tight binding preferably at a unique location o Useful fluorescence properties o No effect on activity - Covalent and non-covalent probes used - very wide range of chemical properties and spectroscopic properties - Used to label macromolecules specifically to investigate their action - Fluorescein (ex = 495 nm / em = 521 nm) - ITC is reactive towards nucleophiles, including amine and thiol groups in proteins. - 1-anilinonaphthalene-8-sulfonate (ANS) - can bind to hydrophobic pocket in a protein - Mention DNA intercalating dyes, e.g. ethidium bromide and SYBRSafe, as examples of non-covalently bound probes

Applications of FRET; In Vivo Fluorescence Microscopy

- Different types of fluorescent protein - Other fluorescent proteins with different spectra have been developed (Blue, Cyan, Yellow, Red) [excitation peaks] - Significant autofluorescence in cells which you can't avoid and will affect your ability to see the fluorescence form at least some of these fluorophores, tendency to work towards longer wavelengths because out of florescence is frequently much worse in this part of the spectrum. - Common FRET pairs: BFP/GFP, CFP/YFP, GFP/RFP, GFP/mCherry - Can be visualised in living cells using fluorescence microscope

Sixth power law

- Efficiency of FRET (E) depends on: [the sixth power law] o overlap of the spectra o separation between D and A o flexibility and movement of D and A (free- movement means probability of non-radiative energy transfer between two dipoles not affected by orientation as on average they'll sample all orientations to get non radiative transfer). - R is the separation between D and A - Ro is a characteristic of the D/A pair (distance where E = 0.5)

Fluorescence of tryptophan

- Emitted light (fluorescence) is lower energy than exciting light - This means that it is at lower frequency (n) or longer wavelength (l) - Remember: E = h n and l = c / n - This example demonstrates that re-emitted light (fluorescence) has a longer wavelength. - Difference in lamda max for excitation and emission. - Excitation and emission spectra for tryptophan in polar environment at lambda max 280nm it loses energy when it moves from the excited state to ground remitting radiation (has longer wavelength - 348nm), hence emitting radiation of longer wavelength so less energy.

Strokes shift for a fluorophore;

- Energy absorbed by a fluorophore is greater than the energy re-emitted as fluorescence (light) - the lmax value of the re-emitted light is larger - Sir George Gabriel Stokes first described this phenomenon in 1852 - the effect is now called the Stokes shift - Defined as the distance in nanometers between the most intense absorption band and the most intense fluorescence emission band (lambda max absorption - emission) - Energy losses and the accompanying Stokes shift are general features of fluorophores in solution - the shifts can be enhanced due to solvent effects, excited-state reactions, complex formation and/or energy transfer to acceptor fluorophore (aka FRET) - Changes in i show changes in energy level between the ground state and the first excited state. - You should be able to predict what the Morse diagrams might look like if a change in the Stokes shift occurs.

Excitation and emission spectra;

- Excitation spectrum (solid line); - vary the excitation monochromator wavelength keep the emission wavelength constant. Emission wavelength for monocramator somewhere near the lambda max emission for the fluorophore record excitation spectrum. - For many fluorophores excitation spectrum is identical to the absorption spectrum of the chromophore, but some finer detail may be missing in the excitation spectrum as you typically make a fluorescence measurement at a much lower concentration of the fluorophore than in an absorption spectrum for the same compound. - Y axis on graph created by converting absorption with beer-lambert law. - Emission spectrum (dashed line); - vary the emission monochromator wavelength keep the excitation wavelength constant - The differences in absorption and fluorescence excitation spectra arise primarily due to the nature in which these spectra are measured/detected in a spectrophotometer and a fluorimeter, respectively. For example, you can measure a fluorescence emission spectrum at a [fluorophore] where the absorbance is barely detectable in a spectrophotometer. You can overcome this issue by increasing the slit width for both monochromators while acquiring the excitation spectrum. However, increasing the slit width of the monochromator makes the light reaching the sample and the detector less spectrally pure. This makes the spectral features that may be observable in a absorption spectrum (recorded in spectrophotometer) feature less prominently in the fluorescence excitation spectrum.

Inner filter effect in fluorescence measurements

- Fluorescence intensity is linearly proportional to the absorbance or optical density of the solution - In a typical fluorimeter, emission is detected at a right angle to the excitation light, and only from near the center of the cuvette - Excitation light will traverse half the cuvette pathlength before emission detection, and the emitted light will traverse half the cuvette pathlength before being detected - Recorded emission intensity will be underestimated as a result of the optical density at the excitation and emission wavelengths - auto-absorption by the solution is called the inner filter effect - Influence of inner filter effect on recorded fluorescence intensity can be estimated using the following equation (if effect present can't calculate just need to dilute: Fcorrected = Frecorded x 10 ^[(ODen + ODex)/2] - F recorded is fluorescence intensity , em is emission at lamba max, ex is lambda max at excitation. - Inner filter effect can induce shifts in the emission maximum and a decrease in the fluorescence intensities observed. - If 10^[(ODem + ODex)/2] term ≈ 1, then correction is unnecessary. - (Right) is high concentration left lower, in first curvette as theres so much flurophore that all the excitation light is being absorbed and readmitted as fluorescence before excitation is able to get to the centre. Hence if too high of concentration affect your ability to make a measurement - need low enough conc so that the inner filter effect is essentially minimal of nonexistant.

Fluorescence spectroscopy

- Fluorescence occurs when photon absorbed by a chromophore is re-emitted with lower energy. - A chromophore is a group (often organic) that absorbs visible or UV radiation - not all chromophores show fluorescence - Why is fluorescence important in studying biological systems; o Sensitive to environment (in vitro) § Conformation § Binding sites § Flexibility § Rotation § Intermolecular distances (FRET) o Readily observed at low concentrations (in vivo) § Visualising processes in living cells § Molecular interactions (FRET, BiFC) § Cell trafficking § Green Fluorescent Protein (GFP) § and variants (YFP, CFP, RFP)

Florescent amino acids

- Fluorescence of most proteins is dominated by the Trp component (assuming they contain this amino acid) due to its high quantum yield and higher molar extinction coefficient at 280 nm efficiently absorbing light at this wavelength. (if protein contains all trp affects it the most). Tyrosine and phenylalanine absorb less light so fluoresce less. - Tyrosine has a quantum yield similar to that of tryptophan, but its emission spectrum is more narrowly distributed on the wavelength scale. This gives the impression of a higher quantum yield for tyrosine. - Molar extinction coefficient of tryptophan enables effective measurement of fluorescence emission at lower [tryptophan].

Fluorescence polarisation;

- Fluorimeter modified with polarizer just after the monochromator - Fluorescence polarization only reports on macromolecular rotation while the fluorophore is in the excited electronic state, i.e. it depends on the lifetime of the excited state (nanoseconds - useful as also timescale for rotational tumbling proteins) - As the protein tumbles/rotates change the orientation of the transition dipole which will then determine the orientation of the polarisation of the emitted light from the fluorophore. - Monitor the polarisation of the emitted light at angles that are either alignes with the polarisation of the excitation light or orthogonal (90degrees) to the excitation light to tell you about the ability of the protein to tumble/rotate in solution. - The excitation transition dipole changes orientation as the chromophore diffuses/rotates in solution. When the excitation dipole is aligned to the electric field vector of the excitation light (theta = 0˚ or 180˚) the absorption of the chromophore is at a maximum. The emitted light (fluorescence in this case) has the same polarisation as the emission transition dipole when the fluorophore is re-emitting. The emission monochromator selects light based on wavelength, not polarisation. You have to add polarisers to your instrument to select for a certain polarisation, e.g. in fluorescence anisotropy.

Instrument - fluorimeter

- Fluorimeter vs spectrophotometer; in addition to excitation monochromator which both have you have an emission monochromator, so not only selecting wavelengths of light that you excite with but also able to select the wavelengths of light that's emitted and ultimately reaches your detector. Second major difference is you typically monitor florescence at 90 degrees to the path of the excitation light but with spectrophotometer you have the detector straight ahead. As spectrophotometer measures absorption need to know how much light gets through the sample, but in fluorimeter measures emitted fluorescence which from solution will radiate at 360degrees out of the curvette so can measure at any angle - but 90 is a good design and wont effect passing light through. - Vary slit width of both the excitation and the emission micrometres which will increase and decrease the amount of light respectively.

FRET

- Förster Resonance Energy Transfer (FRET) is another form of non-radiative decay which involves a long-range dipole-dipole interaction between adjacent donor and acceptor fluorophores. FRET is distance-dependent on the nanometer scale. - Fluorescence lifetime (t) can be expressed in terms of relative rates of radiative (kr) and non-radiative (knr) decay for the excited state: 1/t ≈ kr + knr - where knr = kintrinsic + kenvironmental + kdynamic quench + kstatic quench

Distance dependant of FRET:

- Homopolymer of L-prolyl will form a rigid helix structure, from crystallography know you can measure the length of how that poly paorlin helix changes with the addition of each L-prolyl residue so have ruler of the crystallographic determined disatcne in the end for your polypropylene helix with a certain number of alpro purple residues. Then if you attach at each end your acceptor and your donor fluorophores then measure the fret efficiency as a function of the known distance for your poly prolin helix can calibrate the transfer efficiency the e value for a given distance. - Number (n) of Pro residues was varied - Experimental points (◦) for different values of n - Line is theoretical 6th power dependence - Ro for the dansyl/naphthyl pair is 34 Å - Can be used to study interactions, or movement in the range of 24 to 44 Å

Fluorescence polarisation of anisotropy (upside down T means perpendicular)

- If a fluorescently-labelled macromolecule is immobile on a time-scale comparable to the lifetime of the fluorescent group, then the degree of fluorescence anisotropy or polarisation will remain high - If a labelled macromolecule is immobile on a timescale comparable to the lifetime of fluorescent group, the degree of fluorescence and anisotropy/polarisation will remain high as the fluorophore is attached to something tumbling slowly meaning essentially most of the light that's readmitted will have a polarisation similar/same to polarisation of excitation light. But if mobile/tumbling then the fluorescence will become depoloarised of isotropic - so two different orientations about equivelant. - If the macromolecule is mobile on this time-scale, then the fluorescence will become depolarised or isotropic

Fluorescent intensity; quantum yield and quenching;

- If a molecule is being quenched its effective quantum field has been decreased. - Fluorescence lifetime of excited state is on the order of nanoseconds. - Fluorescence lifetime can be expressed in terms of relative rates of radiative (kr) and non-radiative (knr) decay of the excited state. - kr = 1/t0 (only radiative decay) - kr + knr = 1/t - kintrinsic = decay rate due to natural tendency of electrons to lose energy which depends on specific molecular structure (a more rigid chemical structure is less likely to lose energy through bond rotation) - kenvironmental = decay rate due to frequency of molecular collisions (temperature-dependent), and the size and polarity of the colliding species (polar molecules will interact more readily with excited state electrons) - kdynamic quench = decay rate due to transient collision with quencher (depends on [quencher]) - kstatic quench = decay rate due to association of quencher with fluorophore to form equilibrium complex

Two types of fluorescent probes;

- Intrinsic probes - part of the natural molecule - Intrinsic fluorophores in proteins; o Tryptophan (Trp, W) o Tyrosine (Tyr, Y) o Phenylalanine (Phe, F) - Common nucleic acid bases are not fluorescent - some rare bases in t-RNAs are fluorescent. - Extrinsic probes - non-natural fluorophores, added or attached covalently to protein/nucleic acid.

Fluorescence Anisotropy to Probe Protein-Nucleic Acid Interactions

- Looks at protein DNA interactions as you can very easily purchase olingo nucleotides which are modified at a specific position with a fluorescent group, then anneal the nucleotides to form your duplex, creating the binding site for the potential binding partner/protein. - So this species will tumble very quickly so depolarisation with isotropic fluorescence and then attached to the protein and is stabilise with less depolarisation becomes more anisotropic.

In vivo fluorescence microscopy using fluorescent proteins;

- Martin Chalfie, Osamu Shimomura and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry for their discovery and development of GFP. MW ≈ 27 kDa - Compare to fluorescein GFP (cloned from jellyfish) is very large it's like adding another domain to a protein, so need to check that the fusion of the GFP doesn't affect function. - With GFP once the beta barrel structure forms in the cell it doesn't fluoresce immediately. - The post-translational modification of GFP takes a certain amount of time (3 residues need to form the conjugated cyclic structure - needs oxygen to complete) and is referred to as the "maturation time" for the fluorescent protein. This can take minutes to hours depending on the fluorescent protein, i.e. fluorescent proteins are not immediately fluorescent upon folding into their final 3D structure.

Quantum yield;

- Not all fluorescent molecules are equally powerful fluorophores - Quantum yield (Q) is the probability that an absorbed photon will be re-emitted as fluorescent light at a given condition (e.g. Q varies with temperature) - Q = (number of photons re-emitted)/ (number of photons absorbed) - Q must lie between 0 and 1 - I = intensity of the excitation of the light - c = concentration of fluorophore - Q = quantum yield - Amount of fluorescence ≈ I c Q - Organic dyes often used as they have a higher quantity closer to 1. - Can't increase the concentration of fluorophore over a very wide range due to inner filter effect.

Interpretation of intrinsic protein fluorescence;

- Polarity o Polar (water) wavelength is higher so fluorescence is less intense while, non polar (buried in protein) has a lower wavelength hence higher fluorescence. Environment affects intensity. - Quenchers; o Some species (Cs+, I-, acrylamide) will quench Trp fluorescence, but only if they directly collide with the fluorophore when it's in the excited state (collisional quenching) o When fluorophore is in an excited state it'll cause it to lose energy and decay to the ground state in a non-radiative manner. So essentially quenches the radiative decay and leading to non-radiative decay. Quite useful as with tryptophan can measure protein fluorescence in the presence and absent of quenchers to find how many trytophens are solvent accessible/exposed and able to collide with the quencher. If quenching is occurring you'd see a decrease in fluorescence o Used to assess whether Trp residues are internal or external in folded protein o External fluorophores are quenched o Collisional quenching by Cs+ ion, I- (iodide) ion, or acrylamide is also known as dynamic quenching. o kdynamic quench = decay rate due to transient collision with quencher (depends on [quencher])

Instrumental spectral bandwidth;

- Spectral band width depends on the slit width of the monochromator - Increasing the slit width means that there is more light (good), but the light is less spectrally "pure" (bad) - slit lets more wavelengths of light through - Decrease slit width drops overall intensity which might be harder to make meaurements with. - Spectral band width defined as the band of wavelengths in the central half of the wavelengths (light) passed by the monochromator - Diffraction grating separates wavelengths - have grooves which you can change to give better control on how it hits the sample.

In Vivo Applications of Fluorescent Proteins: conformational changes, interactions and co-localisation

- Yeast cell membrane visualised by membrane proteins fused with RFP and GFP fluorescent markers. Imposition of light from both of markers results in yellow colour. - Bimolecular fluorescence complementation (BiFC) - fuse two target proteins half a beta barrel for GFP, so the two halves of the beta barrel come together forming the competent fluorescent protein. Shows interaction between X and Y which is useful as it can probe for interactions in the cell between two partners in the cell to see their interaction, but this reaction is irreversible - Can do FRET where you have more than one acceptor - do 3 colour FRET with three different target proteins where you want to see if they form a complex. Excite CFP to form a complex with YFP, see some emission from CFP but mostly YFP, then form tertiary complex with RFP see emission from all. Look at more complex interactions in the cell. - Non radiative energy transfer between these different acceptors but if you have GFP na dRFP attached to two separate proteins which didn't form a complex you wouldn't see any FRET but might see a colour which demonstrates co-localisation of those two proteins in the same cellular compartment (plasma membrane). Membrane protein fused GFP and a separate protein that's fused RFP, GFP fused proteins appear green while RFP appear red with both proteins present get an intermediate yellow/orange - shows two protein type labels are colocalized into the same cellular compartment.

study 2

Review the concept of an excitation transition dipole and how its orientation relative to the electric field vector determines how much of the incident electromagnetic energy is absorbed Review how rotation of a fluorophore-labelled macromolecule while the fluorophore is in the excited state affects both the orientation of the emission transition dipole and the polarisation of the fluorescence emission Review the relationship between the molecular weight of a macromolecule and its rotational correlation time (aka its tumbling time) Why do you always fluorescently label the interacting partner with the smaller molecular weight when designing a fluorescence anisotropy experiment to measure a binding interaction? Why is FRET considered a type of quenching for the donor fluorophore? Can you draw out the chemical mechanism whereby the post-translational modification of the T65, Y66 and G67 residues in GFP yields the mature fluorophore?

- Fluorescent protein domain genes can be fused to genes of interest;

o One per protein for molecular interaction - intermolecular FRET o Both ends of one protein for intramolecular (conformational) changes - intramolecular FRET - Mention size of fluorescent proteins relative to extrinsic small molecule probes. - Only get a measurable long-lived FRET signal if your two proteins are truly bound together - if only passing signal too brief to measure. - FRET pairs: BFP/GFP, eCFP/eYFP, eGFP/TagRFP, eGFP/mCherry