Intro to structural biology - GPCRs Flashcards
What type of structures are there for every protein, and what physiochemical factors determine it
Primary, secondary, tertiary, quaternary
Hydrogen bonds, ionic bonds, hydrophobic interactions, and disulphide bonds determine the secondary, tertiary and quaternary structure of the proteins.
Protein folding and its regulation
Protein folding occurs spontaneously after production in the ribosome. They form domains that are self-stabilising and fold independently.
There are chaperones in cells that reduce misfiling and trapped intermediates.
Proteolysis of adhesion GPCRs
Adhesion receptors contain a GPCR auto proteolysis-inducing domain (GAIN). This is very important in tissue development.
Its EC N-terminus can dissociate and recognise other things within the cell. Then the GAIN self-cleaves and dissociates from the GPCR. Thought to have role in signalling and activation of adhesion GPCRs.
X-ray crystallography methodology
A saturated protein solution is slowly evaporated until protein crystals precipitate.
This is difficult for GPCRs as they have to remain in the membrane. To get around this, artificial membranes can be used, or a detergent can be used to get rid of the membrane.
Using an X-ray beam, the diffraction of rays leave a pattern on a detection device, representing the amplitude of electron density using a Fourier transformation. Converts the diffraction patterns mathematically to the electron density map.
Resolution of X-ray crystallography and factors in improving the methodology.
Resolution is a measure of certainty for the electron density map generated. >4 angstrom is bad - only secondary structure. 0.5-1.5 can determine the side chains confidently.
Proteins can be engineered to improve their crystallography. Removal of N-terminus increases crystallisation chances. Homogeny of the sample can be improved by removing glycosylation sites. The conformation can be locked if it is crystalised in complex with a ligand. Thermostability can be improved with addition of a fusion partner, antibody fragment, or making a chimera.
XFEL methodology
X-ray free-electron laser. Can be used with microcrystalline.
It produces short bright pulses of X-rays. It limits the radiation damage, with only one shot of x-ray per crystal. The crystals are kept at room temperature.
The protein dynamics can be studied with this method.
Cryo-EM methodology
Developed to be now at a useable resolution. It is more forgiving in looking at dynamic sections of the protein than X-ray crystallography is.
A pure protein sample is applied to a copper grid and frozen. A transmission electron microscope shoots electrons through it, and the scattered electrons are detected. This is used to produce a 2D projection in different orientations. Hundreds of thousands of these images are compiled to make a 3D reconstruction.
Due to the damaging effects of the electrons on the proteins, this causes noise - reducing resolution. More proteins being used aids this.
NMR methodology
Utilises quantum mechanics - nuclear spin.
A magnetic field causes them to oscillate and this can be detected.
Radiowaves are also used to induce resonance, and this will transfer to nearby atoms.
Needs C13, N15, or H1 for this method.
An experiment where an A2AR modified with F19 used NMR to identify the 3 active states and its 2 inactive states.
Computational models overview
Can simulate the physical movements of atoms and molecules by calculating their potential energies and forces between the particles.
It is used for refinement of structures, protein-protein and protein-ligand interactions, as well as looking at homology modelling.
Newer softwares are getting progressively more accurate at modelling in silico
