Slide 2 Rational Drug Discovery PC session Protein sequence
analysis Biocomputing Primary etc structure X-ray crystallography
Structural genomics Homology modelling Protein Structure QSAR
History Objectives Limitations Statistics Steric Electrostatics
Hydrophobic PC sessions Molecular modelling Theory Drug structure
Drug conformation Docking De Novo ligand design PC sessions FBDD
Lead compound Physiological Biochemical Chemical (prodrugs)
Targeting and delivery Slide 3 3D considerations Protein structure
X-ray crystallography NMR Homology modelling Forming quality
crystals The resolution Solving the structure New for 2010: FBDD
replaces 3D QSAR (Comfa) Slide 4 atoms X-rays 6. Protein structure
6.1. Introduction QSAR most useful when no structure for the target
when structure of target protein is known, molecular modelling more
useful (PC activities are divided between structure known and
structure not known) FBDD is heavily dependent on structure for
designing ways to grow the fragment. Resolution for light to view
an object is about /2. Wavelength, (visible) is about 4000 - 7000
Compare to C-C bond length of 1.5 (X-rays) 20 Slide 5 6.3. X-ray
crystallography Requires a single crystal so scattered radiation
from each atom is in phase A major difficulty in X-ray
crystallography is obtaining crystals which diffract For myoglobin
(1st protein structure, 1957), add solution of ammonium sulphate to
concentrated solution of proteins crystals Some proteins
crystallise easily, some dont trial and error, vary solvent, source
of protein, temperature 10 years for thymidylate synthase (TS)
Membrane proteins (e.g. GPCRs, ion channels, transporters) are the
target for the majority of drugs but very few crystallised Lysozyme
crystals http://crystal.uah.edu/ ~carter/protein/protein.htm A
diffraction pattern Slide 6 6.4. Diffraction apparatus
Illustrations: (above left) liquid nitrogen is used to freeze the
crystal which allows for increased reliability of information
gathered from testing. The area detector, which collects the
diffracted x-rays once they pass through the crystal, is the black
plate located behind the nitrogen stream Not examinable Slide 7
6.5. Recombinant DNA plasmids can be Used to produce a specific
protein (right); robots produce crystals (below) Not examinable
Slide 8 6.7. Detection Film: collects several reflections (spots)
at once - inefficient Scinillation counters collect one reflection
at a time Note X-ray damage to crystal limits time available to
collect data as image gets foggy Area detectors collect several
reflections at once A good structure may require over 100 000
reflections A low resolution structure may be misleading - see
below More powerful X-ray beam (e.g. from a synchrotron as at
Daresbury, Cheshire) can be used to study very large proteins e.g.
virus 8500 kdaltons, 240 protein subunits Slide 9 3 2 1.2 125 K 293
K 6.8. Resolution and temperature The quality and reliability of
the structure depends on the resolution. Generally, below 2 is
good; 1.2 is excellent. From:
http://www-structure.llnl.gov/xray/101index.html at 4 can see the
chain at 2 can see the sidechains at 1 all atoms and solvent
visible Slide 10 6.9. Resolution depends on crystal quality Good
quality crystals but they only diffract to low resolution The
further back the X-rays scatter (i.e. the bigger ), the better the
resolution. 40 Slide 11 6.9. Not a serious slide - but it does show
the difficulty in producing good crystals Perfect crystals disrupt
their formation. The generation of perfect crystals can sometimes
be the limiting factor in determining a protein's structure. are
difficult to achieve on Earth. Gravity and turbulence in the
atmosphere Scientists are now looking at conducting protein
crystallization experiments on the International Space Station. By
eliminating variables such as gravity, crystals are able to form
slower and more precise in space. NASA, in association with the
University of Alabama in Huntsville and the University of
California in Irvine, have been working on the development of the
equipment and the procedure needed to produce protein crystals in
space. Not examinable Slide 12 Crystallization tricks 2 -adrenergic
receptor co-crystallized with lysozyme (red) as lysozyme forms
crystals easily but membrane proteins do not 1 -adrenergic receptor
thermostabilized (binds 50% of ligand at a higher temp) by 6
mutations so that it crystallizes more easily. Slide 13 The active
GPCR structure The 2 -adrenergic receptor (yellow) in complex with
its heterotrimeric G- protein (Gs) (alpha blue, beta cyan and gamma
green). Lysozyme (red) is attached to the N-terminus and a nanobody
(a small camel antobody) is shown in red binding to the G-protein).
Lysozyme and the antibody aid crystallization. Slide 14 6.10.
Solving the structure Calculate the electron density (using
computers) from the diffraction pattern (we dont need to know how)
Fit the amino acids one by one into the electron density contours
(e.g. using computer graphics) to do this need to know the amino
acid sequence (automatic degradation of protein or from cDNA) In
the illustration, the 5-atom imidazole ring from the amino acid
histidine is adjusted from an incorrect location (panel A) to a
correct location (panel B). Slide 15 6.10. Solving the structure
(continued) Structures can be refined using molecular mechanics and
molecular dynamics. Scattering of X-rays is proportional to
electron density so hydrogen atoms are not visible. Cannot
distinguish -CO 2 - from -COOH, -CO 2 - from CONH 2 water molecules
from Na + ions Why is this a problem? 6.10.1. Neutron diffraction
No free radicals generated - no crystal damage, neutrons are
scattered by nuclei, not electrons so H-atoms can be observed.
Disadvantage? Need nuclear reactor! It is a different exercise to
design a drug to bind to CO 2 - rather than COOH or CONH 2 Slide 16
6.11. Modelling protein structure Some out of date statistics:
protein sequences available protein structures available Probably
there is no structure for the protein of interest Need to predict
the structure from sequence 6.11.1. Modelling protein structure by
homology Proteins exist in families with similar 3D structures
(e.g. TIM barrel): i.e. the same pattern of -helices, -sheets,
loops many identical residues at corresponding positions Main
differences in loops which connect important parts 2 proteins show
homology if there is similarity in their sequences 45 5 000 000 30
000 Slide 17 6.11.2. Modelling proteins by homology Align
sequences, preferably in a multiple sequence alignment Display
co-ordinates of known structure Alter sequence of known to that of
unknown make insertions or deletions in the loops building the
loops may be guided by loops of similar structure in the protein
structure databank Adjust orientation of side chains to remove
steric clashes Use molecular mechanics/molecular dynamics to refine
structure Does the structure we have modelled explain the known
experimental facts? If not, adjust accordingly This method works
well if the similarity is high. See PC activity. If there is no
related structure, use structure prediction methods? Slide 18 6.12.
Protein structure by NMR 6.12.1. Nuclear Overhauser effect (NOE)
Change in intensity of one peak when another is irradiated is
called an NOE There is a single absorption frequency for transition
E - E + since most of the molecules lie in lower energy state.
Apply intense radiation corresponding to the transition frequency
for one type of proton - saturate the NMR peak - change spin
population so equal number in two levels The nuclei interact with
neighbouring nuclei and change their spin populations so intensity
changes Effect is proportional to 1/r 6. Advantages: Gives distance
information on protons close together Distance Geometry can be used
to turn distance information into 3D structure Works for proteins
that dont crystalize Disadvantages Need powerful NMR > 500 MHz
Difficulty in assigning peaks Currently works only for small
proteins
