DNA/Protein structure-function analysis and prediction Lecture 12: DNA/RNA structure.
DNA/Protein structure-function analysis and prediction
description
Transcript of DNA/Protein structure-function analysis and prediction
1
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction(Titia Sixma, NKI)
– Electron Microscopy/Diffraction(Titia Sixma, NKI)
– NMR Spectroscopy(Lorna Smith, Oxford)
– Other Spectroscopic methods
2
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Spectroscopy: The whole spectrum
3
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
4
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Structure determination method X-ray crystallography
Purified protein
Crystal
X-ray Diffraction
Electron density
3D structureBiological interpretation
Crystallization
Phase problem
5
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Protein crystals• Regular arrays of protein molecules
• ‘Wet’: 20-80% solvent• Few crystal contacts
• Protein crystals contain active protein• Enzyme turnover• Ligand binding
Example of crystal packing
6
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Examples of crystal packing
2 Glycoprotein I~90% solvent (extremely high!)
Acetylcholinesterase~68% solvent
7
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Problematic proteins• Multiple domains
• Similarly, floppy ends may hamper crystallization: change construct
• Membrane proteins
• Glycoproteins
Flexible
Lipid bilayer
hydrophilic
hydrophilic
hydrophobic
Flexible and heterogeneous!!
8
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Experimental set-up• Options for wavelength:
– monochromatic, polychromatic – variable wavelength
Liq.N2 gas stream
X-ray source
detector
goniometer
beam stop
9
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Diffraction imageDiffraction image
Water ring
Diffuse scattering (from the fibre loop)
reciprocal lattice reciprocal lattice (this case hexagonal)(this case hexagonal)
Beam stop
Increasing resolution
Direct beam
ReflectionsReflections ( (h,k,lh,k,l) ) withwith I( I(h,k,lh,k,l))
10
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
The rules for diffraction: Bragg’s law
• Scattered X-rays reinforce each other only when Bragg’s law holds:
Bragg’s law: 2dhkl sin = n
11
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Phase Problem• If phases hkl and structure factor F(hkl) known:
• compute the electron density (x,y,z)
– In the electron density build the atomic 3D model
• However, the phases hkl are unknown !
hklhkl
hkl
lzkyhxiihklFV
lzkyhxihklFV
zyx
)}(2exp{exp)(1
)}(2exp{)(1
),,(
12
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
FH, HFK, K
FH, KFK, H
How important are these phases ??• Fourier transform photo’s
of Karle (top left) and Hauptman (top right) (two crystallography pioneers)
• Combine amplitudes FK with phase H and inverse-fourier transform
• Combine amplitudes FH with phase K and inverse-fourier transform
(Taken from: Randy J. Read)
13
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
How can we solve the Phase Problem ?• Direct Methods
– small molecules and small proteins– needs atomic resolution data (d < 1.2 Å) !
• Difference method using heavy atoms– multiple isomorphous replacement (MIR)– anomalous scattering (AS)– combinations (SIRAS,MIRAS)
• Difference method using variable wavelength– multiple-wavelength anomalous diffraction (MAD)
• Using a homologous structure– molecular replacement
14
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Building a protein model• Find structural elements:
-helices, -strands• Fit amino-acid sequence
15
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Building a protein model• Find structural elements:
-helices, -strands• Fit amino-acid sequence
16
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Effects of resolution on electron density
Note: map calculated with perfect phases
d = 4 Å
17
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
d = 3 Å
Effects of resolution on electron density
Note: map calculated with perfect phases
18
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
d = 2 Å
Effects of resolution on electron density
Note: map calculated with perfect phases
19
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
d = 1 Å
Effects of resolution on electron density
Note: map calculated with perfect phases
20
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Refinement process
• Bad phases poor electron density map
errors in the protein model
• Interpretation of the electron density map improved model
improved phases improved map
even better model
… iterative process of refinement
21
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Validation• Free R-factor (cross validation)
– Number of parameters/observations
• Ramachandran plot • Chemically likely (WhatCheck)
– Hydrophobic inside, hydrophilic outside
– Binding sites of ligands, metals, ions
– Hydrogen-bonds satisfied– Chemistry in order
• Final B-factor values
22
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
23
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Electron microscopy• Single particle
– Low resolution, not really atomic– Less purity of protein, more transient state analysis
• Two-dimensional crystals– Suited to membrane proteins
• Fibres– Acetylcholine receptor– Muscles, kinesins and tubulin
• Preserve protein by– Negative stain (envelope only)– Freezing in vitreous ice (Cryo-EM, true density maps)
• High resolution possible but difficult to achieve– For large complexes: Combine with X-ray models
24
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Electron diffraction from 2D crystals: Nicotinic Acetylcholine Receptor
Unwin et al, 2005
G-protein coupled receptors
25
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Electron diffraction: near atomic resolution• Structure of the alpha beta tubulin dimer by electron
crystallography. Nogales E, Wolf SG, Downing KH.Nature 1998 391 199-203
26
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Select particles Sort into classes
Average Reconstruct 3D image
Single particle Electron Microscopy
27
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006 Leiman et al. Cell. 2004 Aug 20;118(4):419-29
Cryo EM reconstruction:Tail of bacteriophage T4
28
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
29
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
1D NMR spectrum of hen lysozyme (129 residues)
• Too much overlap in 1D: 2D
• 1H-1H• 1H-15N• 1H-13C
30
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
N
C
C
N
C
C
H
O
N
H
O
H C H
R H
HHC
Amino acid spin system
C
O
Amino acid spin system
Step 1: Identification of amino acid spin systems
31
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Val
Val
Thr
Thr
2D COSY spectrum of peptide in D2O
32
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
N
C
C
N
C
C
H
O
N
H
O
H
C H
R H
HHC
NH(i)-NH(i+1) NOE
H(i)-NH(i+1) NOE
H(i)-NH(i+1) NOE
NH(i)-NH(i+1) NOE
Step 2: Sequential assignment
33
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Gly
Gly
AspAsn
Asp
Phe
ThrSer
Leu
Val
2D NOESY spectrum
• Peptide sequence (N-terminal NH not observed)• Arg-Gly-Asp-Val-Asn-Ser-Leu-Phe-Asp-Thr-Gly
34
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Nitroreductase Dimer: 217 residues• Too much
overlap in 2D: 3D
• 1H-1H-15N• 1H-13C-15N
35
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Structural information from NMR: NOEs• For macromolecules such as proteins:
– Initial build up of NOE intensity 1/r6
– Between protons that are < 5Å apart
36
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
O
N
H
O
N
H
R
H
O
N
O
N
H
O
R
HN
H
O
N
H
R
H
H
H
R
O
N
H
H
R
R
H
R
H
O
N
H
OH
R
H
R
NOEs in -helices• NH-NH(i,i+1) 2.8Å• H-NH(i,i+3)3.4Å• H-H(i,i+3) 2.5-4.4Å
• Cytochrome c552 3D 1H-15N NOESY
– H-NH(i,i+3)– resides 38-47– form -helix
37
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
NN
NN
R
O R
O R
OH
H
H
H
R
OH H
H H
NN
NN
N
R
OR
OR
O H
H
H
H
R
OHH
HH
O
H
NOEs in -strands• H-NH(i,j) 2.3Å• H-NH(i,j) 3.2Å
38
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Long(er) range NOEs • Provide information about
– Packing of amino acid side chains – Fold of the protein
• NOEs observed for CH of Trp 28 in hen lysozyme
39
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
N
C
C
N
C
C
H
O
N
H
O
H C H
R H
HHC
C
O
Spin-spin coupling constants• Fine structure in COSY cross peaks• For proteins 3J(HN.H) useful:
– Probes main chain torsion angle
• Hen lysozyme:
2
3
4
5
6
7
8
9
10
0 20 40 60 80 100 120 140
Co
up
ling
co
nst
an
t (H
z)
Residue number
40
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Structural information from NMR3. Hydrogen exchange rates
• Dissolve protein in D2O and record series of spectra
– Backbone NH (1H) exchange with water (2H)• Slow exchange for NH groups
– In hydrogen bonds– Buried in core of protein
After 20 mins After 68 hours 1H-15N HSQC spectra for SPH15 in D2O
41
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Structural information from NMR• NOEs
– 1H - 2H 1.8-2.5Å (strong)– 2H - 40HN 1.8-5.0Å (weak)– 3H - 88H2 1.8-5.0Å (weak)– 3H - 55H 1.8-3.5Å (medium)
• Spin-spin coupling constants– 1C-2N-2C-2C -120+/-40°– 4C-5N-5C-5C -60+/- 30°– 5C-6N-6C-6C -60+/- 30°
• Hydrogen exchange rates– 10HN-6CO 1.3-2.3Å 10N-6CO 2.3-3.3Å– 11HN-7CO 1.3-2.3Å 11N-7CO 2.3-3.3Å
42
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
NMR structure determination: hen lysozyme
• 129 residues– ~1000 heavy atoms– ~800 protons
• NMR data set– 1632 distance restraints– 110 torsion restraints– 60 H-bond restraints
• 80 structures calculated• 30 low energy
structures used
0
2000
4000
6000
8000
1 10 4
1.2 10 4
10 20 30 40 50 60 70
Tot
al e
nerg
y
Structure number
43
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Solution Structure Ensemble• Disorder in NMR ensemble
– lack of data ?
– or protein dynamics ?
44
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
DNA/Protein structure-function analysis and prediction
• Protein Structure Determination:
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
45
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Ultrafast Protein Spectroscopy• Structure-sensitive technique• State of protein and substrate
– Redox state– Protonation– Elektronen
• Follow reactions in real-time• Why ultrafast spectroscopy?
– Molecular movement: time scales of 10 fs – 1 ps (10-15 – 10-12 s)
– O H stretching frequency: ~3500 cm-1 (~ 10-14 s)• Transfer or movement of proton, electron or C-atom
46
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
1750 1700 1650 1600
-2
0
2
mO
D
Wavenumber (cm-1)
Chl*/Chl in THF Pheo*/Pheo in THFketo
ester
Keto and ester C=O in Chlorophyll a, Pheophytin a are redox and environmental probes
Excited state difference spectra of Chl and Pheo
47
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
X – C – O – H
O – XOω1
ω2
ω1’
ω2’
Why is vibrational spectroscopy sensitive to structure?
48
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
H-bond response during the photocycle
1780 1760 1740 1720 1700 1680-1.0
-0.5
0.0
0.5
mO
D
Wavenumber (cm-1)
2 ps, excited state 700 ps, I0 inf, I1
1740 1720 1700 1680 1660 1640
-1
0
1
2
mO
D
Wavenumber (cm-1)
1.5 ps, excited state 800 ps, I0 inf, I1
S
NHOO
O HO
H
O
O
H
NH
H2N NH2
Cys69
Thr50
Tyr42
Glu46
+
Arg52
Replace Glu with Gln whichdonates weaker H-bond
weaker stronger
broken
49
Str
uctu
re-F
un
cti
on
An
aly
sis
17 Jan 2006
Summary
Method Pro Con
X-ray crystallography
High resolution
No size limits
Easy addition of ligands
Crystals required
Phase problem
Electron microscopy /
diffraction
Single particles possible
Well suited to membrane proteins
Labour intensive
High resolution difficult
NMR spectroscopy
In solution
Dynamic information possible
Resolution variable
Limited size (< ~30kDa)
Other spectroscopy
(Very) high time resolution (ps!)
Very sensitive
Technically extremely complex
Limited applicability
Limited information