Transcript outside the cell

Bi 1 Lecture 3
Thursday, March 30, 2006
What is a Receptor?
Receptors and Ion Channels as Examples of Proteins
1
receptor
a molecule on the cell surface or in the cell interior that
has an affinity for a specific molecule (the ligand).
Latin,
“to tie”
Most drug receptors are proteins.
Greek, “first”
2
shortest: 9
longest: 5500
“peptide”
or
amide bonds
20 types
side chains
link the
“backbone”
or
“main chain”
or
“a-carbons”
Little Alberts Figure 2-22
© Garland publishing
3
Proteins contain a few structural motifs:
a helices
(Swiss-prot viewer must be
installed on your computer)
http://www.its.caltech.edu/~leste
r/Bi-1/alpha-helixalphabetical.pdb
b sheets
http://www.its.caltech.edu/~lest
er/Bi-1/beta-sheetantiparallel.pdb
Hide side chains
Show H-bonds and distances
Show ribbons & arrows
Show side chains
Show Van der Waals radii
4
Most drug receptors are membrane proteins
Outside the cell
nicotinic
acetylcholine
receptor
nicotine,
another agonist
natural ligand
(agonist)
~ 100 Å
= 10 nm
Membrane = lipid bilayer
Inside the cell = cytosol
(view in ~1995)
5
Overall topology of the nicotinic acetylcholine receptor
(view in ~2000)
5 subunits
each subunit has 4 a-helices
in the membrane
(20 membrane helices total)
outside the cell:
Binding Region
Little Alberts figure 12-42
© Garland publishing
6
The acetylcholine binding protein (AChBP) from a snail,
discovered in 2001, strongly resembles the binding region
(Swiss-prot viewer must be
installed on your computer)
5 subunits
Little Alberts figure 12-42
© Garland publishing
http://www.its.caltech.edu/~lester/Bi1/AChBP+Carb-5mer.pdb
Color by chain
Show 2 subunits,
Chains,
Ribbons
7
The AChBP binding site occupied by an acetylcholine analog (2004)
http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb
http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb
8
Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)
~ 2200
amino acids
in 5 chains
(“subunits”),
Binding
region
MW
~ 2.5 x 106
Membrane
region
Colored by
secondary
structure
Colored by
subunit
(chain)
Cytosolic
region
http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9
9
How the binding of agonist (acetylcholine or nicotine)
might open the channel: June 2003 view
Ligand-binding
region
M1
M2
M3
M4
10
Most drug receptors are membrane proteins
Some drugs compete with
nicotine or acetylcholine
Some drugs bind on the axis
membrane
region
Nicotinic acetylcholine receptor
11
All I really need to know about life
I learned in Bi 1
1. If you want a job done right, get a protein
Protein
Lecture #
Ligand-gated Ion Channels
3 (today)
Pumps and transporters
5, 13
Motors
10
G protein-coupled receptors and G proteins
12
Enzymes
13, 15
DNA-binding proteins
18
RNA polymerase, ribosome
18
Cystic Fibrosis Transmembrane Regulator
20
Rhodopsin
26
12
Protein structure prediction:
An important 21st-century problem
Want to test your own skill at predicting protein structure?
Then enter “Critical Assessment of Techniques for Structure Prediction”
or CASP 7
http://predictioncenter.org/
Winners earn an automatic “A+” in Bi1 (retroactively, if appropriate)
13
Protein Folding vs. “Inverse Folding” = Computational Protein Design
Protein Folding
(no degeneracy)
Set of All
Structures
Inverse Folding
(large degeneracy)
Individual
amino acids
Set of All
Sequences
Several ways to
make an arch
14
Bi 1 Cameo by Professor Pamela J. Bjorkman
http://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&response=Detail_Person.html&-layout=all_fields&person_id=29067&-search
X-ray Crystallography
Crystal
Growth
X-ray Data
Electron
Density
Protein
Model
15
X-ray crystallography
• Why X-rays?
Right wavelength to resolve atoms
• Why crystals?
Immobilize protein, enhance weak signal from scattering
• What is a protein crystal?
Large solvent pathways (20-80% solvent)
Same density as cytoplasm
Enzymes active in crystals
• Are crystal structures valid compared with solution structures?
Usually -- Compare NMR and X-ray structures
Structures correlate with biological function
Multiple crystal forms look same -- small effects of packing
16
Overview of imaging
No lens to refocus
X-rays, so must
understand
reciprocal space and
diffraction
Diffraction:
Scattering followed
by interference
17
Bragg’s law
Consider simultaneous reflection of a large number of x-rays.
See diffraction maximum in direction q only if diffracted waves are in phase.
Path difference (2dsinq) must represent an integral number of wavelengths to get
constructive interference.
18
Learned two things from Bragg’s Law
• sinq = nl/2 x 1/d
Low angle: large interplanar spacing
High angle: small interplanar spacing
Since sinq  1/d, structures with large interplanar
spacings (d) will have diffraction patterns with small
spacings and vice-versa.
• Repeating unit in real space (crystal) --> diffraction
maxima and minima
19
Same molecular transform sampled by different lattices
a) Molecular transform
b) Lattice
c) Convolution of
lattice and transform
d - f ) Same molecular transform sampled by different lattices
Modified from Lipson & Taylor, 1964 20
Resolution
An inverse Fourier
transform (FT)
including all of the
high angle
information gives
back the original
image.
An inverse FT
including only the
low angle
information gives
back a low
resolution view of
Mickey.
From Harburn, Taylor, Wellbery,
An Atlas of Optical Transforms 21
The electron density equation and the phase problem
1
(xyz)  
V h
 | F(hkl) | exp[( 2i(hk  ky  lz )  ia
k
Can measure this
|F(hkl)|=I1/2
hkl
]
l
Can’t measure this
• There are experimental methods for
determining the phase for each reflection hkl.
22
X-ray detection
• Film (relic of the past)
• Diffractometers (almost relic
of past, but used for small
molecules)
• Multiwire detectors (almost
relic of past)
• Phosphorimager detectors
(R-AXIS, MAR)
• CCD detectors
23
Synchrotron x-ray sources
e-
• High-intensity x-ray emitted
by charged particles
accelerated in a curved path
• X-ray wavelength in range of
0.5 - 2 Å (from E=hn=hc/l)
• Is tunable!!
Radiation emitted by
accelerating charged particle
tangent to path of circle
24