Protein spectroscopy and dynamics

1. Protein spectroscopy and dynamics Vibrational spectroscopy Time-resolved spectroscopy Hemoglobin Myoglobin Enzymes Protein Folding

2. Dynamics in Proteins • Dynamics consist of: • Protein relaxation in response to - ligand/substrate binding - electron transfer • Protein folding. - cyclic compared to b-sheet peptides - unfolded - molten globule - folded • Time-resolved vibrational spectroscopy is a tool for investigation of structural changes.

3. Vibrational Spectroscopy Quantum theory Normal modes Infrared absorption Raman scattering

4. Classical approach: harmonic approximation Differences with QM approach: The solution is oscillatory. Any energy is possible. Q

5. Quantum theory of vibration Harmonic approximation Energy is quantized v is the quantum number Allowed transitions v’ ® v + 1, v’ ® v - 1 Q

6. The bonding electronic state gives rise to a potential energy surface for the nuclear motion Harmonic approximation

7. There is a potential energy surface that corresponds to each electronic state of the molecule The shift in the nuclear displacement arises from the fact that the bond length increases in the s* state compared to the s state. We will show that the overlap of the vibra- -tional wave functions is key to understanding the shape of absorption bands.

8. There are 3N-6 vibrational degrees of freedom in a molecule with N atoms Three degrees of freedom are required for translation. Three degrees of freedom are required for rotation. For example, in H2O there are 9 total degrees of freedom and 3 vibrational degrees of freedom. In C6H6 there are 36 degrees of freedom and 30 vibrational degrees of freedom. Exception: In linear molecules there are only 2 rotational degrees of freedom and therefore the number of vibrations is 3N - 5.

9. The vibrational degrees of freedom can be expressed as normal modes. All normal modes have the same form for the harmonic oscillator wavefunction and differ only in the force constant k and mass m. The total wavefunction is a product of normal modes. The total nuclear wavefunction for water is c1c2c3. The normal mode wavefunctions of water correspond to the symmetric stretch, bend, and asymmetric stretch. These are linear combinations of the stretching and bending internal coordinates of H2O.

10. Normal modes of water In water vapor n1» n3, but symmetries are different, G1 ¹ G3. (G is the symmetry) However, the third overtone of 1 has the same symmetry as the combination band G1 G1 G1 = G1 G3G3 . Strong anharmonic coupling leads to strong overtones at 11,032 and 10,613 cm-1.

11. Comparison of harmonic and anharmonic potentials

12. Frequency shift due to molecular interactions Hydrogen bonding lowers O-H force constant and H-O-H bending force constant. vapor ® liquid n1 3825 ® 3657 n2 1654 ® 1595 n3 3935 ® 3756 The intermolecular hydrogen bonding stretching mode is difficult to observe.

13. VibrationalTransitions

14. Transition dipoles In order for infrared light to be absorbed the polarization must be aligned with the direction of the transition moment. For a vibrational mode this is determined by the directional change in the dipole moment. This is shown below for the bending mode of H2O.

15. Transition dipoles The change in ground state dipole moment during vibration interacts with light. The first term is static and does not contribute to the transition. Calling the vibrational wave- functions ci the transition moment is:

16. Dipole derivatives The vibrational wavefunctions ci are Gaussians, thus the transition moment for transition from vibrational state 0 to vibrational state 1 is: The transition dipole moment is proportional to the dipole derivative. This is true for any normal mode of vibration (i.e. harmonic).

17. Absorption of infrared radiation leads to vibrational transitions v = 0

18. Absorption of infrared radiation leads to vibrational transitions v = 1 v = 0

19. The selection rule for vibrational transitions is Dv = ±1 v = 2 v = 1 v = 0

20. Analysis of isotope effects Vibrational spectra are analyzed within the harmonic approximation. Reduced mass Classical harmonic oscillator equation

21. Raman spectroscopy Goal: Study vibrational frequencies of the heme and the axial ligands in order to obtain information on the coupling of protein motion and electrostatics with the heme iron

22. Resonance Raman spectrum is obtained by a laser light scattering experiment Detector Lens Spectrograph Sample Laser Inelastic light scattering produces a frequency shift. There is exchange of energy between the vibrations of the molecule and the incident photon.

23. Resonance Raman is a two photon process Incident photon from a laser. Scattered photon has an energy shift. The difference is because the molecule is left in an excited vibrational state. hn

24. The iron in heme is the binding site for oxygen and peroxide Heme is iron protoporphyrin IX. Functional aspects in Mb O ||| O

25. The iron in heme is the binding site for oxygen and peroxide Heme is iron protoporphyrin IX. Functional aspects in Mb 1. Discrimination against CO binding. O ||| C

26. The iron in heme is the binding site for oxygen and peroxide Heme is iron protoporphyrin IX. Functional aspects in Mb 1. Discrimination against CO binding. 2. O2 is the physiologically relevant ligand, but it can oxidize iron (autooxidation). 3+

27. Porphine orbitals eg eg a2u a1u

28. The four orbital model is used to represent the highest occupied and lowest unoccupied MOs of porphyrins The two highest occupied orbitals (a1u,a2u) are nearly equal in energy. The eg orbitals are equal in energy. Transitions occur from: a1u®eg and a2u ® eg. M1

29. The transitions from ground state p orbitalsa1u and a2u to excited state p* orbitals egcan mix by configuration interaction Two electronic transitions are observed. One is very strong (B or Soret) and the other is weak (Q). The transition moments are: MB = M1 + M2 MQ = M1 - M2»0 M1 M2

30. Absorption spectra for MbCO and deoxy Mb Q Band Soret Band

31. Resonance Raman spectrum for excitation of heme Soret band Soret Band B Band Excitation Laser Q Band Raman spectrum

32. Soret (B) band Resonance Raman spectra of MbCO and Deoxy Mb n8

33. B band Resonance Raman spectra of MbCO and Deoxy Mb

34. Hemoglobin Time scale for the R-T switch The trigger mechanism

35. The cooperative R - T switch Hemoglobin is composed of two a and two b subunits whose structure s resemble myoglobin. Eaton et al. Nature Struct. Biol. 1999, 6, 351

36. The frequency of the iron-histidine vibration shows strain in T state The comparison of photolyzed HbCO in the R state and the equilibrium T state. Hb*CO at 10 ns Fe-His = 230 cm-1 Deoxy Hb Fe-His = 216 cm -1 The lower frequency indicates weaker bonding interaction and coupling to bending modes. lexc = 435 nm Fe-His Hb*CO 10 ns R-state Deoxy Hb T-state

37. The heme iron center moves out of the heme plane and the porphyrin macrocycle domes upon deligation of CO CO is photolyzed Fe displacement Planar Heme Domed Heme

38. The ligation of CO changes the spin state of the heme iron S = 0 S = 2 Low spin Fe(II) High spin Fe(II)

39. The motion of the F-helix tugs on the proximal histidine and introduces strain The frequency lowering in the T state arises from weaker Fe-His ligation and from anharmonic coupling introduced by the bent conformation of the proximal histidine.

40. Time-resolved resonance Raman can follow the R - T structure change Time evolution Hb*CO 10 ns 100 ns 400 ns 1 ms 8 ms 15 ms 40 ms 60 ms 120 ms Deoxy Hb Strain is introduced in stages as intersubunit contacts are made. Based on the x-ray data it was proposed that the iron displacement from the heme plane is a trigger for the conformational changes. 200 210 220 230 240 Raman Shift (cm-1) Scott and Friedman JACS 1984, 106, 5877

41. Ultrafast resonance Raman spectroscopy shows that heme doming occurs in »1 ps Equilibrium HbCO Difference spectra obtained by subtraction of the red spectrum from spectra obtained at the time delays shown. The evidence suggests that heme iron displacement is an ultrafast process that is independent of viscosity. Franzen and Martin Nature Structural Biology 1994, 1, 230

42. Dehaloperoxidase: The First Enzymatically Active Globin NC State University

43. DHP oxidizes tribromophenol DHP + DBQ + H2O DHP + TBP + H2O2

44. Many Peroxidases belong to the Cytochrome c Peroxidase family PDB: 1A2F Cytochrome c Peroxidase (CCP) Class: All a proteins Superfamily: Heme peroxidases Family: CCP-like Goodin and McCree Scripps Institute PDB: 2ATJ Horseradish Peroxidase (HRP) Class: All a proteins Superfamily: Heme peroxidases Family: CCP-like Hendrickson et al. Biochemistry (1998) 37, 8054

45. Dehaloperoxidase is a peroxidase that belongs to the globin family PDB: 1A6G Myoglobin (Mb) Class: All a proteins Superfamily: Globin-like Family: Globins Vojetchovsky, Berendzen, Schlichting PDB: 1EW6 Dehaloperoxidase (DHP) Class: All a proteins Superfamily: Globin-like Family: Globins Lebioda et al. J.Biol.Chem. 275 18712 (2000)

46. Amphitrite ornata ~1 cm DHP is the coelomic hemoglobin

47. Comparison of DHP and Mb Structures Mb DHP Superimpose hemes

48. Overlay of active sites Mb DHP

49. Functional questions Where is the pull? Mb DHP Where is the push?

50. Dehaloperoxidase looks like Mb, but dehalogenates halophenols Franzen et al., JACS (1998), 120, 4658-4661