2. What spectroscopy for? By using different parts of the electromagnetic spectrum (different types of light) we can “see” separate details.

3. A Z X Y A =AmplitudeV = frequency λ = Wavelength Phase Electro-magnetic radiation

4. Wavelength Frequency Energy Loss of light Production of light Intensity of diffracted beam Absorption Emission Scattering/Diffraction SourceSampleDetector

5. + = i.e two waves in phase with the same amplitude and wavelength produce a wave that has twice the amplitude, but the same wavelength + = Two waves exactly out of phase with each other cancel each other out.

6. The Fequency (v) & wavelength (λ) of a wave are related by: v = c/λ (C = 3 x 10 8 m.s -1 ) Frequency can be converted to energy (e) by: e = hv (h = 6.63 x 10 -24 J.s ) Scales 1 nm = 10 -7 CM = 10 Å

7. Energy = hc λ

8. H C The circles show energy levels - representing increasing distances from the nucleus. The further out the energy level the higher its energy. Energy  Bonding electron  bonding electron n non- bonding electron  *bonding electron  * bonding electron In organic molecules, the energies of orbitals increase in the order  <  < n <  * <  * S p Carbon electronic configuration

9. What happens when EM radiation hits an object? Energy from a light source interacts with protein molecules in several ways. At an atomic bonding level we see promotion of electrons to higher energy levels. On a molecular level we see absorption and emission. Transition between energy levels is not just confined to electrons; chemical bonds can have a variety of vibrational energy levels & atoms connected together by covalent bonds can rotate relative to each other.

10. e e e e e e e e Transition A transition occurs when the energy of a molecule changes from one state to another - e.g. from ground to first exicted state. Such transitions can be shown on a potential energy diagram Absorption occurs when radiation causes an increase in energy of the system with which it interacts. Emission occurs when radiation is produced by a system during a transition from a higher energy level back to a lower one.

11. The frequency of radiation required to promote a transition between 2 energy staes (E1 to E2)  E = (E2 -E1) =hv E1 E2 E3 Energy E4 Ground The total energy of a molecule is the sum of distinct reservoirs of energy: Translational, vibrational, rotational, electron/nuclear spin orientation states Ground state = lowest energy, E1, E2,…etc are excited states The type of radiation used causes different types of transitions

12. 1 10 3 10 2 10 10 5 10 4 Microwave Infrared visible ultraviolet X-rays Rotational transitions Vibrational transitions Electronic transitions (outer shell) Electronic transitions (inner shell) Energy increases Diffraction

13. Fluorescence Spectroscopy Some chromophores are quite rigid & inflexible thus have a limited range of vibrational energy levels. Often the vibrational energies of the excited & ground states do not overlap so it is not possible for them to return to the ground state by losing energy as heat. In fluorescence spectroscopy, energy is lost by radiative transistion. Ground state Excited state energy Interatomic distance hvhv Fluorescence

14. A portion of the energy absorbed is re-emitted as light. The emitted light is always of longer wavelength (lower energy). We can quantitate fluorescence by the quantum yield, Q Q = Number of photons emitted Number of photons absorbed Under a given set of conditions, Q will have a fixed value for a particular fluorophore, with a maximum possible value of 1. In Fluorescence spectroscopy

16. Light source Monochromators Io λ1 Transmitted light Detector Cuvette containing Sample Emitted light λ2 Intrinsic fluorophores in proteins include Trp, Tyr & Phe. Trp & Tyr give stronger spectra than Phe, & Tyr is frequently quenched as a result of proton transfer in the excited state. The bases of DNA nucleotides & of some co-factors (e.g. NAD) are also intrinsic fluorophores, although they produce weak spectra.

17. 1 2 3 S0S0 S 1’ S1S1 1 Excitation: A photon of energy hv (EX) is absorbed by the flourophore, creating an excited electronic singlet state (S1’). 2 The fluorophore undergoes conformational change & interacts with it’s molecular environment. S1’ energy is partially dissipated, to give a relaxed singlet state (S1) from which fluorescence emission originates. Not all molecules excited by stage 1 return to the ground state (S0) by flourescence emission: quenching, energy transfer and intersystem crossing can also lead to depopulation of S1. 3 A photon of energy hv (EM) is emitted, returning the fluorophore to the ground state S0. The wavelength of emission is longer and the energy is lower

18. Fluorescence Substances which display fluorescence should have: Rigid structures Delocalised electrons (alternate single & double bonds, aromatic rings). Intense U.V absorption bands (π to π* transition, e.g. Trp Em 5700 m -1 cm -1 Short excited state lifetimes (<10 –9 sec). Good overlap between electron orbitals of ground & first excited states. 2 ways of measuring fluorescence: Emission spectrum- excitation λ constant, measure fluorescence intensity of emission against λ, I.e. spectrum of emitted light. Excitation spectrum – measure fluorescence intensity at different excitation λ, similar to absorption spectra.

19. Energy loss & Quenching Only a portion of the light absorbed is emitted as radiation, energy may be lost in vibrational transitions & heat transfer with solvent as well. Two further processes can diminish amount of light energy emitted from the sample. Internal quenching due to intrinsic structural feature e.g. structural rearrangement. External quenching interaction of the excited molecule with another molecule in the sample or absorption of exciting or emitted light by another chromophore in sample. All forms of quenching result in non-radiative loss of energy. Intersystem crossing from excited singlet to excited triplet state. Transition occurs between the singlet ground state (electrons are anti- parallel & paired) to an excited state (electrons are parallel and unpaired). Return to ground state much slower process than fluorescence, = Phosphorescence. Emitted radiation is of an even longer wavelength because the energy difference between the two is small. Resonance energy transfer singlet – singlet energy transfer.

20. Fluorescence intensity (Q) Tryptophan profile Shows a characteristic absorption & fluorescence spectrum 150 100 50 A 200 300 400 2 λmax Max intensity

21. Rules for interpreting Fluorescence spectra of proteins All fluorescence of a protein is due to Trp, Tyr or Phe, unless it contains an extrinsic fluor or fluorescent co-factor. The max of Trp shifts to shorter wavelengths & the intensity of max increases as the polarity of the solvent decreases. If the max is shifted to shorter wavelengths when the protein is in a polar solvent, the Trp must be internal. if max is shifted to shorter wavelengths when the protein is in a non-polar solvent the Trp is on the surface of the protein or conformation changes such that it is brought to the surface. If a quencher (iodide, nitrate ions) quenches Trp fluorescence it must be on the surface of the protein. Failure to do so means its either internal, in a small crevice, or in a highly charged region. Trp fluorescence is quenched by neighbouring protonated acid groups. If a substance binds to a protein & Trp fluorescence is quenched, either there is a conformational change as a result of binding or Trp is in or near the binding site.

22. Fluorescence & Protein folding One of the main techniques used to study protein folding. Folded proteins compact structures - residues adopt highly defined conformations. When a protein unfolds from active (N) to unfolded (U) sidechains become free to rotate around Cα & Cβ bonds & move freely in solution. Trp is normally buried in the hydrophobic core of the protein. When a protein unfolds Trp comes in contact with solvent & may also be able to interact with quenching agents. NU

23. Monitor conversion: native -> unfolded Spectroscopic probes: CD, Fluorescence, FTIR, UV/Vis Spectra change as protein unfolds native denatured Increasing [Urea]

24. Revision points Process is cyclical & only a portion of the energy absorbed is re-emitted as light. Emitted light is of a lower energy & longer wavelength than absorbed energy. Quenching. Emission spectrum = monitor fluorescence intensity at a constant excitation wavelength. Excitation spectrum = monitor fluorescence intensity at different excitation wavelengths holding emission wavelength constant. In proteins, only 2 amino-acids are fluorescent: Trp & Tyr = intrinsic fluorophores. Extrinsic fluorophores e.g. chemical dyes. Uses of fluorescence

25. What is Circular Dichroism? Circular Dichroism (CD) is a type of absorption spectroscopy that can provide information on the structures of many types of biological macromolecules It measures the difference between the absorption of left and right handed circularly-polarized light by proteins. CD is used for; Protein structure determination. Induced structural changes, i.e. pH, heat & solvent. Protein folding/unfolding. Ligand binding Structural aspects of nucleic acids, polysaccharides, peptides, hormones & other small molecules.

26. Plane polarized light E M Direction of propagation Polarizer E vectors

27. Plane & circular polarized light A light source usually consists of a collection of randomly orientated emitters, the emitted light is a collection of waves with all possible orientations of the E vectors. Plane polarized light is is obtained by passing light through a polarizer that transmits light with only a single plane of polarization. i.e. it passes only those components of the E vector that are parallel to the axis of the polarizer. Circular polarized light; The E vectors of two electromagnetic waves are ¼ wavelength out of phase & are perpendicular. The vector that is the sum of the E vectors of the two components rotates so that its tip follows a helical path (dotted line).

32. CD measures the difference between the absorption of left and right handed circularly-polarized light. This is measured as a function of wavelength, & the difference is always very small (<<1/10000 of total). After passing through the sample, the L & R beams have different amplitudes & the combination of the two unequal beams gives elliptically polarized light.Hence, CD measures the ellipicity of the transmitted light (the light that remains that is not absorbed):

33. CD measures the difference between the absorption of left and right handed circularly-polarized light.  A( ) = A R ( )-A L ( ) = [  R  ( ) -  L  ( )]lc or  A( ) =  ( )lc  is the difference in the extinction coefficients typically < 10 M -1 cm -1 cf to typical  around 20 000 M -1 cm -1 So the CD signal is a very small difference between two large originals.

34. CD is only observed at wavelengths where absorbances of R & L components of circularly polarized light are not zero i.e. in absorption bands. The CD arises because of the interaction between different transition dipoles doing the absorption. As this depends on the relative orientation of different groups in space the signal is very sensitive to conformation. So in general  is much more conformation dependent that . We will concentrate on the “electronic CD” of peptides and proteins below 240nm. This region is dominated by the absorption of peptide bond and is sensitive to changes in secondary structure. Can also do CD in near UV (look at trp side chains), visible (cofactors etc.) and IR regions.

35. The peptide bond is inherently asymmetric & is always optically active. Any optical activity from side-chain chromophores is induced & results from interactions with asymmetrical neighbouring groups.

36. wavelength in nm 190200210220230240250 Mean residue ellipicity in deg cm 2 dmol -1 -40000 -20000 0 20000 40000 60000 80000  -helix  -sheet random coil CD signals for different secondary structures These are Fasman standard curves for polylysine in different environments E L – E R > 0 E L – E R < 0

37. Main features of CD spectra of protein 2 ndary structures -ve band (nm)+ve band (nm) α-helix 222 208 192 β-sheet 216195 β-turn 220-230 (weak) 180-190 (strong) 205 L.H polypro II helix 190210-230 weak Random coil 200

38. Applications of CD in structural biology Determination of secondary structure of proteins Investigation of the effect of e.g. drug binding on protein secondary structure Dynamic processes, e.g. protein folding Studies of the effects of environment on protein structure Secondary structure and super-secondary structure of membrane proteins Study of ligand-induced conformational changes Investigations of protein-protein and protein-nucleic acid interactions Fold recognition

39. Why use CD? Simple and quick experiments No extensive preparation Measurements on solution phase Relatively low concentrations/amounts of sample Microsecond time resolution Any size of macromolecule

40. The CD signal for a protein depends on its secondary structure Notice the progressive change in q 222 as the amount of helix increases from chymotrypsin to myoglobin —— chymotrypsin (~all  ) —— lysozyme (mixed  &  ) —— triosephosphate isomerase (mostly  some  ) —— myoglobin (all  )

41. If we measure the CD signal for a protein of unknown structure we can find its proportion of 2ndry structures Fit the unknown curve  u to a combination of standard curves. In the simplest case use the Fasman standards  t = x    + x    + x c  c Vary x , x  and x c to give the best fit of  t to  u while x  + x  + x c = 1.0 Do this by least squares minimization

42. Example fit: myoglobin In this case :  t = x    + x    + x c  c fits best with x  = 80%  x  = 0% x c = 20% agrees well with structure 78% helix, 22% coil

43. Use spectroscopic probe to determine f N and f U Plot signal change (e.g. intensity at one wavelength) vs. denaturant (temp, [urea]). So get f U and f U at different conditions (e.g. [urea]).

44. CD spectra Use as probe for whether protein is folded, unfolded, or misfolded, in response to change in solution conditions, or mutation.

45. Determining free energy of unfolding [urea] vs.  G governed by  G [urea] =  G H2O ° - m[urea]. Extrapolate to 0 to determine  G°. Or use value at some [urea] (  G for mutation, to determine effect of mutation). m gives some measure of change in H-phobic surface area.

47. CD spectra as a measure for protein folding: