Spectroscopies for Structure

1. Spectroscopies for Structure Andy HowardBiology 555: Created 21 April 2014Presented 24 October 2016 and13 November 2018

2. Spectroscopy can be a structural tool • Several types of spectroscopy provide structural information. We’ve already discussed NMR. Today we’ll examine four others.

3. Agenda • Circular dichroism • Theory • Measurements • Application to proteins • Helicity • Melting Curves • Unfolded -> Folded • Electron paramagnetic resonance • Concept • Intrinsic signals • Spin labels • Fluorescence • Generalities • Tyr and trp • Ligands • Exposures • Quenching • Mass spectrometry • Protein MS • Electrospray • MALDI-TOF • Application to proteomics • Is this structural?

4. Circular dichroism • This is a form of spectroscopy that capitalizes on the fact that chiral molecules (including polypeptides and polynucleotides) differentially absorb left- and right-circularly polarized light • The extent to which they do so is wavelength-dependent, and measurements of those wavelength-dependent differences can tell us something about structure

5. Polarization of light Cartoons courtesy Wikipedia • Remember that, with EM radiation, E and B are always perpendicular to one another and perpendicular to the direction of propagation • Linear polarization: E oscillates in only one plane • Circular polarization: direction of E rotates about propagation direction but maintains its magnitude

6. Left- and right- circular polarization • Left-circularly polarized light: the electric vector rotates counter-clockwise • Right-circular polarized light: E rotates clockwise • It’s possible to filter light to separate out the left- and right-circularly polarized components

7. What happens when polarized light interacts with chiral matter? • E causes a linear displacement of charge • B causes a circulation of charge • Together that moves the electron in a helical motion • Achiral molecules: every electron that absorbs more left-circular than right-circular will be offset by its mirror image • Chiral molecules: there may be a differential absorption, and that differential absorption will be wavelength-dependent

8. Quantitation • We measure difference in absorption ΔA between left and right • By Beer’s law ΔA = ClΔε • ClearlyΔε is wavelength-dependent; that’s why these measurements involve spectroscopy.

9. Ellipticity as a rotation angle • Express the difference as the angle associated with this depiction of the difference in transmission • This enables us to express the effect either as a CD value Δε(l) or an ellipticityθ(l) in radians or degrees or centi-degrees

10. Relationship between Δε and [θ] • Intensity is proportional to |E|2 • So θ = (IR½ – IL½) / (IR½ + IL½) • But remember that Beer’s law is really an exponential absorption: I = I0e-q = I0e-Aln10 • But that means IR½ = I0e-(AR/2)ln10 and IL½ = I0e-(AL/2)ln10 • Taylor expansion of the θ equation givesθ (radians) = (ΔA/4)(ln10) orθ (degrees) = (ΔA/4)(ln10) (180/π) • Converting to centidegrees and dividing by concentration gives us [θ] = 100Δε[(ln10)/4] (180/π) • Thus [θ] = 3298.2Δε

11. If you have lots of components rotating things… • If we’re studying a polymer, and many monomers are doing the same kind of differential absorption, we’ll get additive effects on [θ] or Δε • Therefore we may want to measure:mean residue ellipticity = [θ]/m,where m is the number of monomers in the polymer

12. Where would you make these measurements? • The largest amount of data can be derived from ranges of wavelengths where the molecule is absorbing a lot anyway • For biopolymers this is in the ultraviolet range, typically from 190 to 260 nm. • It’s harder to make reliable measurements below 190 nm, largely because oxygen absorbs strongly; even the 190-200 nm range is tricky

13. Using CD on proteins • Proteins are polymers of chiral amino acids • All proteins (except polyGly!) exhibit a CD spectrum • Fingerprint analysis could tell you when the protein is changing conformation… but we should be able to do better than that • It turns out that secondary structural motifs have specific CD spectra—especially α-helices

14. CD spectra for pure secondary structures • Spectra from real proteins: rarely this clean!

15. Structural information • A complex protein structure could be decomposed into a linear combination of the spectra shown in the previous slide • Software packages exist to do that • In practice, separating strands from random coils and disordered structures has limited predictive value • So the primary application is to helicity

16. Practical application I: estimating helicity • Comparing an observed spectrum to an idealized alpha-helical protein spectrum can yield an estimate of the percentage of the structure that is helical • Results correlate well with helix measurements derived from crystal or NMR structures • Doesn’t require crystals or high concentrations or ultra-high purity

17. Results for Vitreoscilla hemoglobin • These structures are 80-90% helical

18. Melting curves • CD spectrum will be temperature-dependent if the structure changes as a function of temperature • CD can be followed to measure unfolding, particularly with helical proteins Vitreoscilla hemoglobin melting curves

19. Structural transitions • Proteins with minimal structure can sometimes be induced to fold into a more regular structure • Via interaction with a ligand • Via protein-protein interaction • These transitions can be readily followed via CD

20. Electron paramagnetic resonance spectroscopy • An unpaired electron will interact with an external magnetic field via Maxwell’s equations • EM radiation will generate an energy difference between low-energy spin state and high-energy spin state for unpaired electron • Microwave electromagnetic fields are the correct energy range for resonating with typical differences between low and high

21. EPR physics

22. EPR signals from proteins • Rate of rotation of a spin influences the resonance behavior of the spin • Tryptophan and tyrosine can harborunpaired electrons under appropriateconditions • Creation of these states can be monitored • Technique is sensitive (low background) • Generally independent of protein size

23. Spin labels • If you covalently or noncovalently attach a ligand that contains an unpaired electron to a protein it will produce a signal • This can be monitored to enable an analysis of the neighborhood of the spin label

24. Nature of the labels • Often the unpaired electron is on a nitrogen atom • Nitroxide (-N=O) is a common instance • Nitroxide often incorporated into a ring (e.g. pyrrolidine) to enable residue-specific reactivity

25. Applications to structure Next several slides from Vanderbilt Univ. structural biology

26. Mobility correlates with position within the protein

27. Multiple labels • If you’ve labeled a protein in more than one place per monomer, you can estimate the distance between the labels from analysis of dipolar coupling between the labels • This works over a narrow range of distances— 1 - 2.5 nm; but that’s a range that’s considerably wider than NMR or XAFS

28. Distance determination in proteins

29. Fluorescence spectroscopy • Fluorescence is emission of a higher-wavelength photon after absorption • Signal tends to be sharp and low-background • Wide applications, some non-structural

30. Fluorescence for structure • Trp, tyr, and phe absorb soft-UV reasonably well • They re-emit at characteristic wavelengths • Those wavelengths and the fluorescent yield are environment dependent, which is what makes this a useful structural tool • Tryptophan is the most important

31. Using trp fluorescence • Fluorescence peak for buried trp is blue-shifted (10-20nm) and usually more intense compared to exposed trp: less dielectric • Quenched by nearby acidic amino acids • Unfolding can be monitored by looking at the fluorescence λmax and (for a specific system) at the intensity

32. Extrinsic fluors • Covalent ligands • Enable probes of micro-environment • Sophisticated chemistries available for attachments to particular groups (primary amines, thiols) • Noncovalent ligands work too • These will be in (boundunbound equilibrium) • Often chosen so that they only fluoresce when bound • Often anionic • Natural and human-produced

33. Green fluorescent protein • Protein from jellyfish • Protein contains ser-tyr-glypost-translationally modifiedto 4-(p-hydroxybenzylidine)-imidazolidin-5-one • Intense absorption @395, 475nm; emits @509 • Can be covalently attached at N or C ends to other proteins to use as a fluorescent tag • Also used as a cell-component tag • Mutants with different spectralproperties are available

34. Mass spectrometry • Direct measurement of mass-to-charge ratios is potentially useful • We can do this either with intact proteins or with proteolytic digests of proteins • Once m/z resolution had reached 0.1% or better, people started yearning to use MS on proteins

35. Built-in problem • MS wasn’t used much on macromolecules until the 1980’s because the macromolecule would fragment when ionized • Two techniques for protecting the protein from fragmentation were developed: • Electrospray Ionization (ESI) • Matrix-Assisted Laser Desorption Ionization (MALDI)

36. Electrospray Ionization • Liquid containing the analyte is dispersed by electrospray into a fine aerosol • You want a lot of the solvent to disappear, so you mix water with volatile organics like methanol • Conductive cosolutes like acetic acid are added to increase conductivity and provide source of ions

37. ESI-MS, continued • The tiny droplets produced in ESI can then be subjected to MS analysis • As originally developed, it was coupled to a single MS instrument; more often it’s now coupled to tandem (MS – MS) equipment

38. MALDI • Sample is mixed with a matrix (organic crystals in acetonitrile or ethanol), applied to a metal plate • Pulsed laser irradiates sample, pulling it off the plate • Analyte is ionized in a hot plume of gases • Ions are accelerated into the MS instrument

39. MALDI-TOF • Typical implementation is a time-of-flight spectrometer, where the instrument records the arrival time of the ions to the detector • Often combined with HPLC so that the individual samples are at least somewhat separated by molecular weight or charge • Can even be used in identifying bacteria

40. Coupling MS with proteolysis • Standard technique in proteomics: • Fragment the protein with a protease • Do ESI-MS or MALDI-MS on the fragments • Consult a library of existing fragment mass spectra and look for correspondence • Can be used on massively complex mixtures • Is this really a structural technique? Sort of…

41. MS protocols

42. Hydrogen exchange • As with NMR and neutron diffraction, we can substitute deuterium for hydrogen in our proteins and try to identify which amide protons have been exchanged • This method has become reasonably practical as a structural technique, partly because software for identifying the exchanged amide protons is getting better

43. Other structural approaches • Chemically cross-link the protein at residues that are close together in space but not in sequence before fragmenting the protein • Laser-induced covalent binding to protein will probe how accessible particular parts of the protein are

44. MS in proteomics