Lecture 10: Structural biology of proteins and the importance of protein structure in health and disease Flashcards
Why does protein structure matter?
Molecular interactions achieve specificity in three ways:
Molecular shape: different molecules fitting together like a 3D jigsaw
Chemical complementarity: positive charges interacting with negative charges, hydrogen bonding potential fulfilled, clusters of hydrophobic moieties
Spatiotemporal overlap: molecules being in the same place at the same time
X-ray crystallography
One of the most common methods of determining protein structure is x-ray crystallography
Small crystals of pure proteins are grown and exposed to an x-ray beam
The way the x-rays are bent (diffracted) by the ordered molecules in the crystal enables us to determine the position of every atom in the protein
x-ray source- protein crystal - x-ray diffraction- electron density- protein structure
NMR spectroscopy
Protein structures can also be solved using nuclear magnetic resonance (NMR)
Protein structures are solved in solution, not a crystal lattice
Proteins must be isotopically labelled (15N, 13C) as natural isotopes are not NMR active
NMR structural determination is only possible for ‘small’ proteins (<~30 kDa)
It is possible to measure protein dynamics
Cryo-electron microscopy (cryoEM)
The most recent method of solving protein structures is transmission cryo-electron microscopy
No crystals and only small amounts of protein are needed
Proteins are deposited on a carbon grid, frozen in vitreous (non-crystalline) ice, and imaged. Thousands of slightly out of focus molecules are selected, averaged and used to calculate the final structure
Most recent cryo-EM structures are at atomic resolution
Loss of function
DF508 in cystic fibrosis transmembrane conductance regulator (CFTR)
CTFR is a transmembrane protein channel which controls the movement of Cl- ions across the cell membrane
Cystic fibrosis is a genetic condition caused by mutation in this channel
This leads to a build-up of sticky mucus in the lungs and digestive tract and increased respiratory infections. The median age of people in UK with CF dying in 2017 was 31 (www.cysticfibrosis.org.uk).
Most common mutation is a deletion of residue 508 (DF508). This leads to a channel which is unstable in the open conformation and which is degraded by cells.
Gain of function
G12 KRAS mutations in cancer
KRAS is a small protein which controls many signalling processes. Active KRAS leads to cell growth and proliferation.
Active KRAS is bound to GTP (guanosine triphosphate)
Inactive KRAS is bound to GDP (guanosine diphosphate)
KRAS catalyses the hydrolysis of GTP to GDP (ie inactivates itself)
Mutations in KRAS lead to reduced hydrolysis (ie increased cell growth) and are found in many cancers.
Common mutations are G12D, G12V, G12R and Q61H
Change in interacting partners
E6V mutation in sickle cell disease
Haemoglobin is a protein in our red blood cells (in our blood) which carries oxygen around the body
Haemoglobin consists of four chains: 2x alpha globin, 2x beta globin
Haemoglobin also binds a non-protein chemical group (a prosthetic group). This part – the haem group – is the part of haemoglobin which binds oxygen.
Sickle cell disease is caused by a single mutation (E6V) in beta globin
This mutation causes the molecules of haemoglobin to stick together, forming long polymer rods
This changes the shape of the red blood cells, which become elongated and clog capilliaries resulting in enormous pain for an affected individual
Drug resistance
Gatekeeper mutations in protein kinases (EGFR)
Epidermal growth factor receptor (EGFR) is a receptor protein which crosses the cell membrane
EGFR is activated when a short peptide on the outside of the cell (called EGF) binds to it
Activation of EGFR leads to cell growth (increased proliferation, migration and adhesion)
Overactive (mis-regulated) EGFR is a driver of cancers, including non small-cell lung cancer (NSCLC)
EGFR inhibitors reduce the activity of EGFR and one – called gefitinib – is a therapy for NSCLC
Sadly, the cancer eventually develops resistance to these inhibitors through a mutation in an amino acid in the drug binding pocket called the gatekeeper residue
Gatekeeper mutations are from a small amino acid to a large amino acid and prevent inhibitor binding
This leads to therapeutic resistance (ie the medicine doesn’t work any more)
There are three main methods used to determine protein structures:
X-ray crystallography
NMR
cryo-electron microscopy (cryo-EM)
Each method of structure determination is based on a different physical principle
Which method is used depends on the size of the protein and what you want to find out
