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Other structural methods
Descripción
(Methods) Protein Form and Function Mapa Mental sobre Other structural methods, creado por sophie_connor el 25/05/2013.
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methods
protein form and function
protein form and function
methods
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Resumen del Recurso
Other structural methods
Crystallography
Understanding function: active sites
Understanding similarities between proteins
Understanding mechanisms
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
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
Techniques for examining protein folding structure
Circular dichroism spectroscopy
Fourier transform infrared spectroscopy (FTIR)
Fluorescence
Hydrogen/deuterium exchange
Electroparamagnetic resonance
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
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
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
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
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
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
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
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)
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
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
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
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
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
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
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
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
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