Lecture 8 Methods of Studying Protein Structure II

Question 1

What is Crystallography?

Answer 1

• Crystallography can reveal the atomic structure of
  macromolecules
• Considering only the technique for X-ray crystallography
• Crystallography requires Crystals and x-ray beams.
• The technique works because crystals have building
  blocks of molecules
• The X-rays interact with the electrons of these
  molecules

Question 2

What is crystallisation?

Answer 2

• Slowly changing the solution environment around

a protein to make it less soluble

Question 3

Whats an example of crystallisation?

Answer 3

• ‘Slow salting out’ is one approach to creating crystals
• Salts are added to a protein
• The right concentration induces the protein to
  crystallise but not precipitate

Question 4

Why is water important in crystalls?

Answer 4

• Crystals are mounted into the X-ray beam
• Water is always present in protein crystals
• They must not dry out

Question 5

What’s the basic set-up for X-ray Crystallography?

Answer 5

• An X-ray source is required
• A laboratory source is usually from a Copper X-ray tube
• Cu is used as a target for electrons
• Produces X-rays with a characteristic wavelength:
• λ = 1.54Å
• Another X-ray source (2 xray sources in total!)
• Synchrotron Radiation
• Electrons accelerated in a ring of magnetics
• Emit electromagnetic radiation = X-rays
  + NOTE: AND other wavelengths (other types of radiation)

Question 6

Describe the basic procedure of X-ray crystallography.

Answer 6

• The X-rays strike the protein crystal
• Some pass straight through
• Some collide with the molecules in the crystal
• They scatter in numerous but specific directions
• This scattering is called elastic
• The total scattering produced is called the Diffraction Pattern
• The diffraction pattern is recorded as a number of spots by an electronic detector
• Each spot is a scattered reflection of X-rays
• Each unique spot has a unique intensity
• Each spot contains information about ALL the atomic positions in the protein molecules
• Maths is used to obtain the atomic positions of the molecules
• The maths technique employed is the Fourier Transform

Question 7

What problem exists with the fourier transform technique?

Answer 7

• Fourier transforms need the phase information for each unique spot
• The position of the crest and trough of the
  scattered wave of each diffraction spot
  relative to the other spots
• In X-ray crystallography this phase information is lost
  + This is known as the phase problem
• Extra techniques need to be used to gain back this
  phase information

Question 8

After collecting the data what is the next step in X-ray crystallography?

Answer 8

• The next stage is to calculate an electron density map
• High density = Where atoms are (high amount of atoms= high amount of electrons)
• Low density = Where atoms are not
• Ultimately a protein structure can be produced
• The amount of detail in this structure depends on the resolution of the structure = How easy it is to distinguish features of atoms/ the level of detail of the structure in the map.

Question 9

What would be visible at different resolutions in an electron density map?

Answer 9

• At 6.0Å resolution = The overall course of the chains only
• Between 2.8Å and 4.0Å resolution = Groups within protein structures can be determined / approximation where side chains might be
• Between 2.0Å and 2.5Å = Reliable positions for the side chains of residues
• Typically protein structures are solved around 2.0Å
• Between 1.0Å and 1.5Å resolution = individual atoms can clearly be seen

Question 10

Does protein conformation change when it becomes crystalline?

Answer 10

• Proteins exist usually in their native conformations when they are crystalline
• Proven when enzymes were shown to be working as crystals
• Protein crystals can be up to 80% water anyway

Question 11

What are some potential issues with X-ray crystallography?

Answer 11

• Structures are NOT dynamic

- Potential for distortion through crystal contacts

Question 12

What does Circular Dichroism Spectroscopy measure?

Answer 12

• Circular dichroism (CD) spectra measure how proteins

interact with circularly polarised light

Question 13

How can CD be used to discern the conformation of a protein?

Answer 13

• Secondary structures in proteins have specific CD features
• The conformation of a protein consists of different amounts of these secondary structure features
• CD can be used to see if a protein is in its native conformation in different solution conditions
• The CD spectrum from a protein is a combination from
  these features:
• CD(Protein) = %CD(α-helix) + %CD(β-Sheet)
  + %CD(β-Turn) + %CD(“Other”)
• CD is a very sensitive technique for noting changes in the percentages of secondary structure
• If a protein CD spectrum in a given solution differs from the spectrum in physiological conditions the protein is no longer in its native conformation

Question 14

What is SRCD?

Answer 14

• An extension of CD :
• Synchrotron Radiation Circular Dichroism (SRCD)
• More powerful light source
• Extended lower wavelength range (light blue area)
• More information from the spectrum data