Other structural methods

Other structural methods

1. Crystallography

   • Understanding function: active sites
   • Understanding similarities between proteins
   • Understanding mechanisms

2. Types of protein structure

   • Not all amenable to crystallography
   • Proteins are able to fold and are dynamic
   • Proteins are able to form large complexes such as vesicles fusing at the neuromuscular junction

3. Techniques for examining 3D structure

   • X-ray crystallography
     • Limitation: needs a soluble proteins that will crystallise and diffract
   • NMR
     • Limitation: needs a soluble protein that is quite small
   • Electron microscopy
     • Limitation: needs a large protein that has a regular structure
   • Atomic force microscopy (AFM)
     • Limitations: in formation obtained
   • X-ray diffraction

4. Techniques for examining protein folding structure

   • Circular dichroism spectroscopy
   • Fourier transform infrared spectroscopy (FTIR)
   • Fluorescence
   • Hydrogen/deuterium exchange
   • Electroparamagnetic resonance

5. Assembling proteins

   • Fibrous
   • Extracellular matrix (elastin, collagen)
   • Cytoskeleton (intermediate filaments, microtubules)
   • Muscle (actin, myosin, troponin, tropomyosin)
   • Amyloid (abnormally assembled protein that accumulates in disease tissue)
   • Complexes
     • Ribosome, proteasome, histones, clathrin
     • F1/F0 ATPase

6. Circular dichroism spectoscopy

   • Differential absorption technique
   • At any given wavelength the absorption of left and right handed circularity polarised light by peptide bond is conformation specific
     • Gives rise to characteristic CD spectra
   • Measured as a function of wavelength
   • Chiral molecules (proteins) absorb left and right handed polarised light to different extents
   • Different secondary structures give different spectra
   • Advantages
     • Small amount of material needed
     • Very quick (30mins)
   • Disadvantages
     • Difficult to interpret mixtures of structure therefore result is an average
     • Limited information about secondary structure
       • FTIR can be used as a complementary method to determine secondary structural elements
   • Near UV CD
     • Provides information about tertiary structure
     • Detects changes in chromophores
     • Can be used to detect folding
     • Aromatic side chains show signals at characteristic wavelengths so can be monitored to follow changes in conformation
     • Can be used to compare protein stability

7. Types of spectroscopy

   • Infrared spectroscopy
     • Measures bond vibration frequencies in a molecule to determine functional groups
   • Mass spectrometry
     • Fragments the molecule and measures the masses
   • Nuclear magnetic resonance
     • Detects signal from hydrogen atoms and can be used to distinguish isomers
   • Ultraviolet spectroscopy
     • Uses electron transitions to determine bonding patterns

8. Molecular vibrations

   • Light is absorbed when radiation frequency= frequency of vibration in the molecule
   • Covalent bonds vibrate only at certain allowable frequencies
     • Associated with types of bonds and movement of atoms
   • Vibrations include stretching and bending

9. Attenuated total reflectance

   • Crystal can be diamond, germanium, zinc or selenium
   • Signal is dependent on sample thickness and whether it is a solid or liquid

10. Fluorescence

    • Measures the environment of chromophores
    • Chromophores are excited and emit at specific wavelengths and can vary depending on the environment
    • Determines protein folding or compare protein folds and conformational changes
    • Dye binding can be used to follow fibrillogenesis

11. FTIR spectroscopy

    • Similar structures can be compared using FTIR
    • Advantages
      • Very little material needed
      • Spectra recorded very quickly
      • Can be used under different conditions to monitor structural changes and differences
      • Similar to CD to allow interpretation of secondary structures compared to standard spectra
    • Disadvantages
      • Difficult to analyse and give detailed strucutural information
      • Artefacts can confuse interpretation
      • Weak spectra from some proteins

12. Atomic force microscopy

    • Measures the height of a protein above the surface in the air or liquid
    • Contact mode
      • cantilever is dragged across the surface and deflection is detected indicating damage
    • Tapping mode
      • Oscillated up and down
    • Non-contact mode
      • Oscillates above the surface (in liquid)

13. Electron microscopy for structural studies

    • Why transmission electron microscopy?
      • Wide range of biochemical conditions
      • Large, complex samples are suitable
      • Can work with less than 1mg protein
      • Crystals and high solubility not required
      • All conformations available for analysis
    • Transmission electron microscope images
      • Images equivalent to those of an x-ray of a person's chest
    • Useful for looking at structures of
      • Macromolecular assemblies
      • Viruses
      • Large particles in different states
      • Fibrous proteins
      • Membrane proteins
    • Carried out under a vacuum to prevent air scatter
    • Lenses are magnetic coils instead of glass
    • How is the image formed?
      • Electrons pass through the sample and are either...
        • Pass through unmodified
        • Scatter inelastically
        • Scatter elastically
        • Reflected back
        • Absorbed
      • Two types of contrast
        • Amplitude contrast indicating loss of electrons
        • Phase constant indicating interference of electrons
      • Electron microscope
        • An electron beam is focused onto the sample using a set of magnets
          • It is then focused onto a detector screen
        • Contained in a vacuum to prevent air and particle influence
        • Proteins don't like being in a vacuum
        • Lenses are magnetic coils to focus the electron beam
        • EM grids are 1mm and are often made of mesh copper with a plastic or carbon layer on top to stop the protein falling through
      • Image must be processed to get an increased signal for the structure
        • Single particle averaging
        • Helical image reconstruction of fibrous proteins
        • Electron crystallography of 2D crystals
    • Negative staining
      • Specimen stained with electron dense material such as a heavy metal
        • Some of the electrons passing through the specimen are scattered by structures stained with electron dense material and appear dark
          • Others pass through parts of the specimen that are not stained to form an image on a phosphophorescent screen and appear light
      • Tells you about the morphology of the fibres but not how the chains are arranged
        • Needs more resolution

14. Cryo-TEM

    • If we want a physiologically relevant image then the sample needs to be in a physiologically relevant environment
    • Protein is in an aqueous environment
      • Protein encased in ice allows different orientations to be seen
      • Not affected by gravity
    • Can observe the shape of the complex and internal structures of organelles
    • Increased resolution allows us to see subunit arrangements
    • Able to detect conformational changes
    • Can visualise secondary structure at high resolution
    • Atomic resolution not possible
    • How does it work?
      • Ice is used to encase protein
      • Electron microscope grid is covered in carbon and protein will find itself into one of the holes
      • Liquid layer will cover protein and when put into liquid ethane surrounded by liquid nitrogen it will be frozen
      • Ice is completely see through
        • You cannot see this ice through the electron microscope
    • Radiation damage
      • EM can cause damage by bombarding a protein with electrons which is very destructive
        • Negative stain protects from radiation damage
      • Cyro-TEM uses low dose of electrons to reduce damage
        • Damage to ice will cause melting
          • Results in low resolution image
    • Averaging noisy images
      • Sum noisy images together to get a clearer image
      • This works because signal is being added together and noise is random
      • Averaging many images of the same macromolecule in the same orientation can lead to increase in the signal to noise ratio
        • Signal is the same in all images but the noise is random
          • Signal is boosted
      • Shared suffering
        • A low electron dose can be used multiple times to produce a high resolution picture
        • Full electron dose would have been lethal

15. Single particle analysis

    • Used to look at the 3D structure of a protein
    • A low image signal means you are unable to see the single molecules
    • Raw images must be added together
      • Orientations compared
        • Reconstruct images to get a 3D image of data
    • How is the information extracted from the image?
      • Determine the orientation of the project with respect to the original image
        • Collect sufficient projections of different orientations to give complete information on the object
          • Generate 3D model
            • Increase signal by averaging many projections together
    • Clathrin cages in vitro
      • Assemble a football like structure
      • From an image you can see how clathrin fits together forming a triskeleton structure

16. Icosahedral viruses

    • Many viruses are arranged in shells of protein subunits with overall icosahedral symmetry
      • The virus is so symmetrical that orientations can be determined using the symmetry
        • E.g. hepatitis B and Ross River
    • Icosahedrons have 20 faces, 6 five fold axes, 10 three fold axes and 15 four fold axes
    • Ross River virus
      • Gives you information about the outside of the protein
      • You can peel off layers and see how the protein shell is constructed
      • Crystal structures of intracellular components
      • Greater resolution image can show secondary structures
    • Hepatitis B virus
      • Electron microscope showing virus capsids in ice
        • Narrowed down to picked out molecules
          • End up with views of the structure that become higher and higher resolution
            • Greater resolution image can show secondary structures

17. Electron crystallography from 2D crystals

    • Some proteins will crystallise in a 2D layer
    • Electron micrograph image is collected
      • Diffraction pattern is calculated
        • Interpretation is complicated as electrons are scattered more effectively than x-rays and may be deflected several times
    • End up with an electron density map

18. Fibrous proteins

    • Structural proteins that provide support, shape or protection
    • Elongated
    • Have a specific sequence
    • E.g. collagen, keratin or silk
    • Electron microscopy and x-ray fibre diffraction can be used to look at them
      • Relies on repetitive structure
    • Do not crystallise because
      • Insouble
      • Heterogenous in length
      • Flexible
    • Have a crystallise structure along the fibre axis

19. Helical arrays

    • Some samples form filament or tubes with helical symmetry
    • Identifying the repeat or lattice allows a 3D model to be generated
    • All orientations of the sample are available
    • Examples: nicotinic acetylcholine receptor, actin, kinesin
    • Can calculate diffraction pattern from the tubular 2D crystal image and projection image
    • Diffraction pattern is an arrangment of layer lines which give information about repeating units along fibre axis
      • Can either be..
        • Crystalline fibre
          • Random orientation of crystallites around the fibre axis
          • A diffraction pattern that has sharp spots on layer lines
        • Non-crystalline fibre
          • Polymer molecules that are not packed into crystals
          • Diffraction can give continuous diffraction rather than sharp spots

20. Fibres generally have helical and cylindrical symmetry

    • Means they form the same way around
    • Can calculate a Fourier transform
      • Helix forms a series of spots
        • Regular repeating pattern forms a line of dots
          • A real fibre would be a combination of the 2
    • Helix can be represented as a series of slits
    • DNA diffraction pattern tells you about the difference between base pairs and that it is helical
    • X-ray fibre diffraction and cryo-electron microscopy combined to give overall picture of fibrous protein structure and how it works
      • Diffraction patterns taken when muscle is relaxed and contracted and compare
        • Crystal structures can look at different parts of the molecule