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Lernmaterialien für AC II an der Universität Wien

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TESTE DEIN WISSEN

Which are the classically used precipitating agents?

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  • Inorganic salts: NaCl, ammonium sulphate, Na citrate
  • Organic solutes: ethanol, isopropanol, butanol, MPD
  • Linear polymers: PEG 
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TESTE DEIN WISSEN

What are enantiomorphic space groups?

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There are 230 combinations for all symmetry operations including mirror and inversion operation as well. Biological macromolecules such as proteins, DNA and RNA structures are however restricted to non-mirror and non-inversion operation, because those operation would otherwise change the chirality of the molecules. The combination of operations of possible operations are the chiral or enantiomorphic space groups, from which we only have 65.

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TESTE DEIN WISSEN

Describe the method MIR: How does it work (theory and praxis)? What does isomorphous mean?

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Theory:

  • Multiple Isomorphous Replacement (MIR)
  • Changing the diffraction pattern by introducing extra scattering matter of known (or readily determined) structure can produce information on phases
  • Basic principle: binding of heavy atom (Hg, Pt, U, Pb, etc) on macromolecule such that the structure of macromolecule does NOT change upon binding to this atom -> Isomorphous
  • Heavy atom derivates show a difference from the native structure in the diffraction pattern, the difference is only in the intensities of the scattered beams.
  • Heavy atoms bind to the functional groups of macromolecules, predominantly on surfaces

Praxis: 

  • Measure the native protein structure factor amplitudes |Fp(hkl)| 
  • Add a heavy atom to the protein and measure the structure factor amplitudes for the heavy atom derivative |Fph(hkl)|
  • From the known position of the heavy atom(s), found by Patterson methods, calculate Fh 
  • From the above construction, the phase αp(hkl) can be calculated for reflection Fp(hkl). But there are two solutions!! So we need more than one derivative to solve the phase problem.
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TESTE DEIN WISSEN

MIR: Which heavy atoms do we use?

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  • Hg, Pt, U, Pb, Au, Th, Re, Os, Ir, Xe
  • Noble Gases (Xe, Kr)
  • Lantanides, iodinated tyrosines
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TESTE DEIN WISSEN

MIR: Why do we need more than one derivative to uniquely solve the phase problem?

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TESTE DEIN WISSEN

From the first calculations, we get two solutions for the phase. Aminoacids are chiral molecules, so it must be only one phase correct. To find which one, we need multiple experiments.

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TESTE DEIN WISSEN

Warum sind Miller Indices Integer?

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TESTE DEIN WISSEN

Because they correspond to Bragg planes, which always have to divide the unit cell edge by an integral number. Otherwise the different unit cells will all diffract out of phase and the waves will cancel out.

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TESTE DEIN WISSEN

Synchrotron? How does it work? What is it used for?

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TESTE DEIN WISSEN

A synchrotron is a machine that accelerates electrons almost to the speed of light. It is used among others for X-ray crystallography. 

  • Electrons circulate in high vacuum at close-to-light speed
  • When they pass a magnetic field they change their velocity and direction, and emit electromagnetic radiation tangentially to their direction

Synchrotron radiation used for structural biology because of its:

- flux: macromolecular crystals are weak diffractors

-high collimation/brillance: small crystals

-tunability: solution of phase problem with MAD or SAS

-time structure of beam: time resolved experiments for study of enzymatic reactions


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TESTE DEIN WISSEN

TF/DSF, what dies are used?

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TESTE DEIN WISSEN

The choice of dye depends on scientific question, protein, detection system, buffers and compounds. 

  • ANS (8-anilinonaphthalene-1-sulfonic acid): for detergent micelles, requires UV excitation 
  • Sypro orange: for globular, soluble proteins
  • CPM: for membrane proteins
Lösung ausblenden
TESTE DEIN WISSEN

TF/ DSF, advantages, disadvantages?

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TESTE DEIN WISSEN
  • Very small quantities of sample protein is enough
  • Low concentration of the protein is needed
  • Reproducible results
  • Correlates with DSC and CD
  • Fast
  • Allows simultaneous screening of multiple conditions

Con: requires compactly folded (globular) proteins, can be difficult to interpret

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TESTE DEIN WISSEN

Hydrophobic effect?

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TESTE DEIN WISSEN

The tendency of nonpolar molecules to self-associate in water rather than to dissolve individually. A nonpolar compound in water disrupts the H-bonding network of water and forces it to re-form around the nonpolar molecule, making a cage around it. The cage is an ordered structure, and is unfavored by the second law of thermodynamics, which stated that spontaneous reactions proceed with an increase in entropy. If the hydrophobic parts aggregate it causes a decreases in order of water, which is entropically favorable.

Lösung ausblenden
TESTE DEIN WISSEN

Microcalorimetry contributions to binding energetics?

Lösung anzeigen
TESTE DEIN WISSEN

Overall binding affinity KD is directly related to dG, the total free binding energy. 

  • dH, enthalpy is indication of changes in hydrogen and van der Waals bonding
  • -TdS entropy is indication of changes in hydrophobic interaction and conformational changes
  • N, stoichiometry indicates the ratio of ligand-to-molecule binding
  • dG = dH-TdS
Lösung ausblenden
TESTE DEIN WISSEN

SEC: What kind of column media/packing do you know and what are their characteristics?

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TESTE DEIN WISSEN
  • Superdex – Spearation MW of 102 to 6 * 105 
  • Superose – Spearation MW of 103 to 5 * 106 
  • Sephacryl– Spearation MW of 103 to 108 

Superdex: Dextran covalently attaced to highly cross-linked agarose. Best for high resolution. 

Superose: Highly cross-linked, agarose-based chromatography medium. 

Sephacryl: Allyl dextran and N,N´-methylene bisacrylamide, cross-linked to form a strong and stable matrix. Sephacryl for bigger molecules and broader range of molecular weight.

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Q:

Which are the classically used precipitating agents?

A:
  • Inorganic salts: NaCl, ammonium sulphate, Na citrate
  • Organic solutes: ethanol, isopropanol, butanol, MPD
  • Linear polymers: PEG 
Q:

What are enantiomorphic space groups?

A:

There are 230 combinations for all symmetry operations including mirror and inversion operation as well. Biological macromolecules such as proteins, DNA and RNA structures are however restricted to non-mirror and non-inversion operation, because those operation would otherwise change the chirality of the molecules. The combination of operations of possible operations are the chiral or enantiomorphic space groups, from which we only have 65.

Q:

Describe the method MIR: How does it work (theory and praxis)? What does isomorphous mean?

A:

Theory:

  • Multiple Isomorphous Replacement (MIR)
  • Changing the diffraction pattern by introducing extra scattering matter of known (or readily determined) structure can produce information on phases
  • Basic principle: binding of heavy atom (Hg, Pt, U, Pb, etc) on macromolecule such that the structure of macromolecule does NOT change upon binding to this atom -> Isomorphous
  • Heavy atom derivates show a difference from the native structure in the diffraction pattern, the difference is only in the intensities of the scattered beams.
  • Heavy atoms bind to the functional groups of macromolecules, predominantly on surfaces

Praxis: 

  • Measure the native protein structure factor amplitudes |Fp(hkl)| 
  • Add a heavy atom to the protein and measure the structure factor amplitudes for the heavy atom derivative |Fph(hkl)|
  • From the known position of the heavy atom(s), found by Patterson methods, calculate Fh 
  • From the above construction, the phase αp(hkl) can be calculated for reflection Fp(hkl). But there are two solutions!! So we need more than one derivative to solve the phase problem.
Q:

MIR: Which heavy atoms do we use?

A:
  • Hg, Pt, U, Pb, Au, Th, Re, Os, Ir, Xe
  • Noble Gases (Xe, Kr)
  • Lantanides, iodinated tyrosines
Q:

MIR: Why do we need more than one derivative to uniquely solve the phase problem?

A:

From the first calculations, we get two solutions for the phase. Aminoacids are chiral molecules, so it must be only one phase correct. To find which one, we need multiple experiments.

Mehr Karteikarten anzeigen
Q:

Warum sind Miller Indices Integer?

A:

Because they correspond to Bragg planes, which always have to divide the unit cell edge by an integral number. Otherwise the different unit cells will all diffract out of phase and the waves will cancel out.

Q:

Synchrotron? How does it work? What is it used for?

A:

A synchrotron is a machine that accelerates electrons almost to the speed of light. It is used among others for X-ray crystallography. 

  • Electrons circulate in high vacuum at close-to-light speed
  • When they pass a magnetic field they change their velocity and direction, and emit electromagnetic radiation tangentially to their direction

Synchrotron radiation used for structural biology because of its:

- flux: macromolecular crystals are weak diffractors

-high collimation/brillance: small crystals

-tunability: solution of phase problem with MAD or SAS

-time structure of beam: time resolved experiments for study of enzymatic reactions


Q:

TF/DSF, what dies are used?

A:

The choice of dye depends on scientific question, protein, detection system, buffers and compounds. 

  • ANS (8-anilinonaphthalene-1-sulfonic acid): for detergent micelles, requires UV excitation 
  • Sypro orange: for globular, soluble proteins
  • CPM: for membrane proteins
Q:

TF/ DSF, advantages, disadvantages?

A:
  • Very small quantities of sample protein is enough
  • Low concentration of the protein is needed
  • Reproducible results
  • Correlates with DSC and CD
  • Fast
  • Allows simultaneous screening of multiple conditions

Con: requires compactly folded (globular) proteins, can be difficult to interpret

Q:

Hydrophobic effect?

A:

The tendency of nonpolar molecules to self-associate in water rather than to dissolve individually. A nonpolar compound in water disrupts the H-bonding network of water and forces it to re-form around the nonpolar molecule, making a cage around it. The cage is an ordered structure, and is unfavored by the second law of thermodynamics, which stated that spontaneous reactions proceed with an increase in entropy. If the hydrophobic parts aggregate it causes a decreases in order of water, which is entropically favorable.

Q:

Microcalorimetry contributions to binding energetics?

A:

Overall binding affinity KD is directly related to dG, the total free binding energy. 

  • dH, enthalpy is indication of changes in hydrogen and van der Waals bonding
  • -TdS entropy is indication of changes in hydrophobic interaction and conformational changes
  • N, stoichiometry indicates the ratio of ligand-to-molecule binding
  • dG = dH-TdS
Q:

SEC: What kind of column media/packing do you know and what are their characteristics?

A:
  • Superdex – Spearation MW of 102 to 6 * 105 
  • Superose – Spearation MW of 103 to 5 * 106 
  • Sephacryl– Spearation MW of 103 to 108 

Superdex: Dextran covalently attaced to highly cross-linked agarose. Best for high resolution. 

Superose: Highly cross-linked, agarose-based chromatography medium. 

Sephacryl: Allyl dextran and N,N´-methylene bisacrylamide, cross-linked to form a strong and stable matrix. Sephacryl for bigger molecules and broader range of molecular weight.

AC II

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