Other Structural Methods

Other Structural Methods

1. Techniques for examining protein structure

   • 3D
     • X-ray crystallography
       • Limitations
         • Must be soluble
         • Must crystallise
         • Must diffract
     • Nuclear magnetic resonance
       • Must be soluble
       • Must be quite small
     • Electron microscopy
       • Must be large enough to see
       • Must have regular structure
       • Advantages
         • Applicable to a wide range of biochemical conditions
           • Large and complex samples
           • All conformations suitable
         • Doesn't need crystals or high solubility
         • <1mg
       • Projection images
         • More like an X-ray than a photo!
       • Applications
         • Macromolecular assemblies
           • Ribosomes
         • Viruses
           • Hep B
         • Large particles, different states
           • AChR
           • GroEL
         • Fibrous proteins
           • Tubulin
           • Muscle
         • Membrane proteins
           • 2D crystals
             • Rhodopsin
       • Sample under vacuum
         • Prevents air scatter
       • Magnetic coil 'lenses'
       • Forming the image
         • Electrons pass through the sample
           • Pass through unmodified
           • Scatter inelastically
           • Scatter elastically
           • Reflected back
           • Absorbed
         • Types of contrast
           • Amplitude
             • Loss of electrons
           • Phase
             • Interference of electrons
               • Retains "which way round" something is
         • Negative staining
           • Stain the specimen with an electron dense material
             • e.g. uranyl acetate
             • Some electrons passing through the specimen are scattered by structures stained with electron dense material
               • Appear dark
               • Others pass through parts that are not stained
                 • Form image on phosphorescent screen
         • Requires processing
           • Single particle averaging
             • Single proteins
             • Protein assemblies
               • Virus assembly and orientation
                 • Icosahedral
                   • Hepatitis B core protein
                   • Ross River virus
                 • Repeating units and symmetry
           • Helical image reconstruction
             • Fibrous proteins
           • Electron crystallography
             • 2D crystals
               • Some proteins crystallise in 2D layer
                 • Bacteriorhodopsin
                 • Collect EM image as usual
                   • Calculate diffraction pattern
                     • DIFFICULT because electrons scattered more effectively than X-rays
                       • Dynamic scattering
         • Resolution
           • 100A
             • Shapes of complexes, internal structure of organelles
           • 3A
             • Atomic
         • Grids
         • Particle suspension
           • Layer of vitreous ice
             • Completely smooth and clear, no crystals
             • Around mesh EM grid which is covered in carbon
         • Radiation damage
           • Bombarding a protein with electrons is destructive
             • Negative staining "protects" the protein
               • Can only use a very low dose of electrons for unstained samples
                 • Very low contrast in the image
         • Noise reduction
           • Averaging many noisy images
             • The signal is the same in all images
               • Noise is random and therefore different
                 • Increase signal-to-noise ratio
             • "shared suffering"
               • Each macromolecule used takes only 1/1024 of the dose required for the final image
                 • Assuming a sum of 1024 images
                 • The full dose would have been lethal to an individual macromolecule
             • More images = better resolution
               • Align 2D images with 3D starting model
               • Projections of different orientations to give complete information on the object
                 • For some proteins, molecules vary. An example is clathrin.
     • Atomic force microscopy
       • Measures height of protein above the surface in air or liquid
         • Resembles a record player
         • E.g. DNA sample + Mg2+ on top of negatively charged mica surface
         • Doesn't accurately measure width!
       • Modes
         • Contact
           • Drag cantilever across surface and detect deflection
             • Risk of damage
         • Tapping
           • Oscillates up and down
         • Non-contact
           • Oscillates in liquid above surface
     • X-ray diffraction
       • Fibre diffraction
         • Limitations
           • Must be fibrous
           • Must diffract
       • Small-angle X-ray scattering
   • Folding
     • Circular dichroism spectroscopy
       • Differential absorption technique
       • At any given wavelength 180-280nm the absorption of left and right handed circularly polarised light by peptide bonds is conformation specific
         • Characteristic spectra dependent on the protein conformation
           • The difference between absorption of left and right handed circularly-polarised light
             • Measured as a function of wavelength
           • Determine protein stability
         • Chiral proteins absorb left- and right-handed polarised light to different extents
           • Optically active
         • Can interpret mixtures of structures or heterogeneous solutions
         • As a protein assembles it may change its secondary structure
           • E.g. alpha-synuclein
       • Advantages
         • Requires less than 100ug material
         • Takes ~30 minutes
       • Disadvantages
         • Difficult to interpret mixtures of structure, if there are different populations in a solution
           • The result is an average
         • Information about secondary structure is limited
           • Can enhance this by combining with FTIR
       • Near-UV
         • Information about tertiary structure
         • Changes in chromophores and disulphides
           • Aromatic side chains show signals at characteristic wavelengths
         • Can detect folding
     • Fourier transform infrared spectroscopy
       • Measures bond vibration frequencies in a molecule
         • Can determine functional groups
         • Light absorption
           • Radiation frequency = frequency of vibrations in molecule
             • Covalent bonds will only vibrate at discrete frequencies
               • Associated with types of bonds, movement of atoms
         • Stretching
           • Symmetric
           • Antisymmetric
         • Bending
       • Attenuated total reflectance
         • Sample above crystal
           • Diamond, germanium, zinc or selenium
         • Signal influences
           • Sample thickness
           • State
       • Advantages
         • Needs only 1-100ug
         • Quick
         • Can monitor structural changes and differences
         • Similar to CD with interpretation of secondary structures
       • Disadvantages
         • Difficult to analyse
           • Limited structural information
           • Artifacts can confuse interpretation
         • Some proteins give weak spectra
     • Fluorescence (ANS)
       • Measure the environment of chromophores
         • Excite and emit at specific wavelengths
           • Varies dependent on environment
         • i.e. are they buried or exposed?
       • Measure and compare protein folds
         • Conformational changes
     • Hydrogen/deuterium exchange
     • Electroparamagnetic resonance
   • Comparing spectroscopic techniques
     • IR
       • Bond vibration
       • Determine functional group
     • Mass
       • Fragment molecule
         • Measure masses of fragments
     • NMR
       • Detects signals from hydrogen atoms
         • Distinguish isomers
     • UV
       • Electron transitions
         • Determine bonding patterns

2. Assembly proteins

   • Fibrous
     • Extracellular matrix
       • Elastin
       • Collagen
     • Cytoskeleton
       • Microtubules
       • Intermediate filaments
     • Muscle
       • Actin
       • Myosin
       • Tropomyosin
       • Troponin
     • Amyloid
   • Complexes
     • Ribosome
     • Proteasome
     • Histones
     • GroEL
     • Clathrin
     • F1/F0 ATPase

3. Applications

   • Fibrous proteins
     • Structural
       • Support
       • Shape
       • Protection
     • Elongated
     • Examples
       • Collagen
       • Keratin
       • Silk
     • Methods
       • EM
       • Fibre diffraction
     • Regular repeating structure
     • Don't crystallise
       • Insoluble
       • Flexible
       • Heterogeneous in length
       • BUT - crystalline order along fibre axis
         • X-ray fibre diffraction
         • EM helical analysis
     • Helical arrays
       • Filaments or tubes with helical symmetry
         • Identify repeat and lattice
           • Full 3D model can be generated
         • All orientations are available
       • Examples
         • NAchR
         • Actin
         • Kinesin
       • EM diffraction
         • Diffraction pattern is a measure of the repetition
           • Shows layer lines
     • Crystalline fibre
       • Random orientation of crystallites around the fibre axis
       • Diffraction cylindrically averaged however has Bragg spots on layer lines
         • Like a crystal rotated 360 degrees
     • Non-crystalline fibres
       • Polymer molecules not packed into crystallites
     • Cylindrical symmetry
       • The pitch is the distance between pattern repeats
     • DNA helix structure determined by fibre diffraction
       • Helix can be simplistically represented as a series of slits
       • Also used for the detection of amyloid
     • Combine X-ray diffraction and cryo-EM to give overall picture of fibrous protein structure
       • Also insight into how it works
       • Example: muscle fibres
       • Can model crystal structures into the electron microscopy maps
     • Understanding a nanomachine
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