enzyme mechanisms b. x-ray methods 1 small angle x-ray scattering (sax) kinetic crystallography –...

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Enzyme mechanismsB. X-ray methods

Small angle X-ray scattering (SAX)

Kinetic crystallography – slow reactions

Kinetic crystallography – Laue method

Kinetic crystallography – freezing techniques

Anomalous dispersion

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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

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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?

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open modelled closed closed

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Complex with pyruvate-P (•)

(1) Fitted with open structure

(2) Fitted with closed model

(3) Fitted with closed structure

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Protein solution without and with UDP-glucosamine

Fitted with open structure

Fitted with closed structure

-substrate

+substrate

fit closed

fit open

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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: ?

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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.

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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.

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Photoactive yellow protein: The photocycle

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Photoactive yellow protein: pG groundstate

chromophore H-bonded to Tyr42

trans double bond,

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Photoactive yellow protein: pR = first excited state at 1- 1.2 ns after excitation

chromophore H-bonded to Tyr42

cis double bond,

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Photoactive yellow protein: pB = second excited state at 2-12 ms after excitation

chromophore H-bonded to Arg52, cis double bond

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Photoactive yellow protein: The photocycle

pG pR pB

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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.

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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.

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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.

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CuZn

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

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Electron density calculated from normal scattering.

‚Anomalous electron density‘ calculated from anomalous scattering at Cu and Zn wavelength.

ZnCu

Zn

Cu

enzyme mechanisms b. x-ray methods 1 small angle x-ray scattering (sax) kinetic crystallography –...

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