1. Enzyme mechanisms B. X-ray methods 1 Small angle X-ray scattering (SAX) Kinetic crystallography – slow reactions Kinetic crystallography – Laue method Kinetic crystallography – freezing techniques Anomalous dispersion

2. Enzyme mechanisms B. X-ray methods 2 Small angle X-ray scattering (SAX): Waves are scattered at objects with which they interfere. The finer the lattice (i.e. small objects), the larger the scattering angle. X-rays (electromagnetic waves) are scattered at objects of molecular size (nm) with variing electron density. direct beam detected signal X-ray

3. Enzyme mechanisms B. X-ray methods 3 Small angle scattering: MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) The enzyme has two domains, which are in a closed conformation (with substrate and inhibitor) or an open conformation (without ligand) – in the crystal structures. Are the differences an artfact of the crystal structure or of relevance for the solution structure?

4. Enzyme mechanisms B. X-ray methods 4 Small angle scattering: MurA open modelled closed closed

5. Enzyme mechanisms B. X-ray methods 5 Small angle scattering: MurA Complex with pyruvate-P () (1)Fitted with open structure (2)Fitted with closed model (3)Fitted with closed structure

6. Enzyme mechanisms B. X-ray methods 6 Small angle scattering: MurA Protein solution without and with UDP-glucosamine Fitted with open structure Fitted with closed structure -substrate +substrate fit closed fit open

7. Enzyme mechanisms B. X-ray methods 7 Kinetic crystallography – slow reactions Remember: L-Haloacid dehalogenase. Trapping of the covalent intermediate by cryocooling (‚freezing‘) during the reaction. Data collection needs to be faster than preparation / soaking / reaction. Data collection: < 10 min at a strong X-ray source for data set, s for single frames, ns - ms for Laue frames. Preparation: s, Soaking: 10-100 s, Reaction: ?

8. Enzyme mechanisms B. X-ray methods 8 Kinetic crystallography – Laue method Normally only single wavelength X-ray light is used for diffraction experiments: ca. 100 frames / data set. With white X-rays ( = 0.8-2 A) only few frames are needed and the intensity is higher: ms / frame.

9. Enzyme mechanisms B. X-ray methods 9 Kinetic crystallography – Laue method Photoactive yellow protein: Light triggers a conformational change in the protein. After a short laser puls (ns), a Laue photograph (ns) is taken. The protein relaxes and the procedure is repeated.

10. Enzyme mechanisms B. X-ray methods 10 Kinetic crystallography – Laue method Photoactive yellow protein: The photocycle

11. Enzyme mechanisms B. X-ray methods 11 Kinetic crystallography – Laue method Photoactive yellow protein: pG groundstate chromophore H-bonded to Tyr42 trans double bond,

12. Enzyme mechanisms B. X-ray methods 12 Kinetic crystallography – Laue method Photoactive yellow protein: pR = first excited state at 1- 1.2 ns after excitation chromophore H-bonded to Tyr42 cis double bond,

13. Enzyme mechanisms B. X-ray methods 13 Kinetic crystallography – Laue method Photoactive yellow protein: pB = second excited state at 2-12 ms after excitation chromophore H-bonded to Arg52, cis double bond

14. Enzyme mechanisms B. X-ray methods 14 Kinetic crystallography – Laue method Photoactive yellow protein: The photocycle pGpRpB

15. Enzyme mechanisms B. X-ray methods 15 Kinetic crystallography – freezing techniques A crystal of a protein-substrate complex is cryocooled. The reaction is started, e.g. by laser. The reaction cannot proceed, because motions are frozen. At increasing temperature further steps may be enabled.

16. Enzyme mechanisms B. X-ray methods 16 Kinetic crystallography – freezing techniques Myoglogin: A crystal with a CO complexed to heme is irradiated with a laser. CO dissociates from the heme. But: due to the low temperature, the CO cannot diffuse out of the binding pocket. At higher temperature the CO can be seen on its way out.

17. Enzyme mechanisms B. X-ray methods 17 Anomalous dispersion: Normally the incident X-ray continues without phaseshift after the scatterer. If the energy is at or above the absorption edge (energy to eject an electron from the atom) of the scatterer, a phaseshift occurs: anomalous scattering. The anomalous scattering is quantified by f‘‘. It is strongly wavelength dependent.

18. Enzyme mechanisms B. X-ray methods 18 Anomalous dispersion: Cu Zn Use this energy to see an effect from Cu and Zn Use this energy to see an effect only from Cu E Only normal scattering

19. Enzyme mechanisms B. X-ray methods 19 Anomalous dispersion: Electron density calculated from normal scattering. ‚Anomalous electron density‘ calculated from anomalous scattering at Cu and Zn wavelength. Zn Cu Zn Cu