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

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

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?

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

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

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

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: s, Reaction: ?

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 ( = A) only few frames are needed and the intensity is higher: ms / frame.

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.

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

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

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

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

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

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.

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.

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.

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

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