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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Enzyme mechanismsB. X-ray methods
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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Enzyme mechanismsB. X-ray methods
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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Enzyme mechanismsB. X-ray methods
Small angle scattering: MurA
open modelled closed closed
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Enzyme mechanismsB. X-ray methods
Small angle scattering: MurA
Complex with pyruvate-P (•)
(1) Fitted with open structure
(2) Fitted with closed model
(3) Fitted with closed structure
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Enzyme mechanismsB. X-ray methods
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
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Enzyme mechanismsB. X-ray methods
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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Enzyme mechanismsB. X-ray methods
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.
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Enzyme mechanismsB. X-ray methods
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.
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Enzyme mechanismsB. X-ray methods
Kinetic crystallography – Laue method
Photoactive yellow protein: The photocycle
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Enzyme mechanismsB. X-ray methods
Kinetic crystallography – Laue method
Photoactive yellow protein: pG groundstate
chromophore H-bonded to Tyr42
trans double bond,
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Enzyme mechanismsB. X-ray methods
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,
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Enzyme mechanismsB. X-ray methods
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
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Enzyme mechanismsB. X-ray methods
Kinetic crystallography – Laue method
Photoactive yellow protein: The photocycle
pG pR pB
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Enzyme mechanismsB. X-ray methods
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.
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Enzyme mechanismsB. X-ray methods
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.
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Enzyme mechanismsB. X-ray methods
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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Enzyme mechanismsB. X-ray methods
Anomalous dispersion:
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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Enzyme mechanismsB. X-ray methods
Anomalous dispersion:
Electron density calculated from normal scattering.
‚Anomalous electron density‘ calculated from anomalous scattering at Cu and Zn wavelength.
ZnCu
Zn
Cu