Experiments and forms of protein structure Flashcards
What role can electron microscopy play in determining protein structure?
It has been helpful for membrane proteins and large macromolecular assemblies
What are the advantages of X-Ray crystallography when determining protein structure?
Size does not matter when determining protein structure. no need to use labelled carbons or nitrogens, is very fast after the crystal has been produced
What are the disadvantages of X-ray crystallography for determining protein structure?
Doesn’t show hydrogen bonds, Proteins are not able to be viewed in solution instead need to be turned into crystals which is a difficult and time consuming process, The fact that crystals are crystallised also results in it being in a fixed conformation so it is unable to visualize protein dynamics
What are the advantages of using NMR to determine protein structure?
Proteins can be made in solution so there is no need to attempt to crystallize them, this results in the protein not being in a fixed position allowing protein dynamics to be determined
What are the disadvantages of using NMR to determine protein structure?
In order to gain information C13 and N15 labelling must occur, the fact that the protein is not crystallised means that a range of possible conformations is produced rather than one certain structure, There is a size limit only very small structures of less than 30kDa can be observed, slower to record data
How are proteins commonly turned into crystals for X-ray crystallography?
A protein solution is placed on a slide which is then placed face down over a a beaker contain a precipitant, to create a seal and allow the protein to crystallise by vapour diffusion
How does x-ray crystallography work?
X-rays are shone through the crystal, and the intensity of the scattered x-rays is measured at each point, this allows calculation of the amplitudes and structure factor giving the final product of an electron density map in which a chemically sensible model can be fitted
How does NMR work?
The atomic spin of nuclei in proteins is measured, this spin changes (resonates when blasted with radio waves), as well as being unique for nuclei in different chemical environments
How can a protein structure be calculated from NMR?
The Nuclear Overhauser effect measurements can be used to identify which atomic nuclei are physically close in space
From this a series of 2D pictures are made which provide a set of constraints on protein structure, from these constraints a family of possible structures can be determined
What is the fold of a protein?
The topology or arrangement in three dimensions of the secondary structural elements
What are the all alpha protein folds?
The four helix bundle where amphipathic helices and solvent shielding is used
Coiled coil interactions
What is an all beta protein fold?
The beta barrel which is common for membrane proteins
What are the common types of non-globular proteins?
Membrane proteins which have a limited number of typologies when compared to globular proteins and are more difficult to study
Fibrous proteins which are made up of unusual repetitive sequences giving remarkable properties
What are coiled coil proteins?
Proteins formed from a unique double alpha helical interaction using a seven amino acid repeat which is mirrored on the other helix
1 and 4 are both hydrophobic which forms a hydrophobic core of 4 residues (2 from each amino acid)
2 amino acids adjacent to hydrophobic core form ionic bonds while the rest perform solvent shielding to protect the hydrophobic core
What is the structure of collagen?
Formed from a simple sequence of glycine-proline-hydroxy proline, which forms a polyporline type II helix which is left-handed
the hydroxy proline here is formed by post translational modification by proline hydroxylase
Three helices with this sequence bind together to form a right-handed super helix, which forms an overlapping, staggered array known as tropocollagen
