Download DNA/Protein structure-function analysis and prediction

Structure-Function Analysis
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
17 Jan 2006
1
Structure-Function Analysis
Spectroscopy: The whole spectrum
17 Jan 2006
2
Structure-Function Analysis
DNA/Protein structure-function
analysis and prediction
• Protein Structure Determination:
17 Jan 2006
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
3
Structure-Function Analysis
Structure determination method X-ray
crystallography
Crystallization
Purified
protein
Phase problem
Crystal
X-ray
Diffraction
Electron
density
Biological interpretation
17 Jan 2006
3D structure
4
Structure-Function Analysis
Protein crystals
• Regular arrays of protein molecules
• ‘Wet’: 20-80% solvent
• Few crystal contacts
• Protein crystals contain active protein
• Enzyme turnover
• Ligand binding
17 Jan 2006
Example of crystal packing
5
Structure-Function Analysis
Examples of crystal packing
Acetylcholinesterase
~68% solvent
17 Jan 2006
2 Glycoprotein I
~90% solvent
(extremely high!)
6
Structure-Function Analysis
Problematic proteins
• Multiple domains
Flexible
• Similarly, floppy ends may
hamper crystallization:
change construct
hydrophilic
• Membrane proteins
Lipid
bilayer
hydrophobic
hydrophilic
• Glycoproteins
Flexible and heterogeneous!!
17 Jan 2006
7
Structure-Function Analysis
Experimental set-up
• Options for wavelength:
– monochromatic, polychromatic
– variable wavelength
Liq.N2 gas stream
X-ray source
beam stop
detector
goniometer
17 Jan 2006
8
Structure-Function Analysis
Diffraction image
Diffuse scattering
(from the fibre loop)
Water ring
Direct beam
Beam stop
reciprocal lattice
(this case hexagonal)
Reflections (h,k,l) with I(h,k,l)
Increasing resolution
17 Jan 2006
9
Structure-Function Analysis
The rules for diffraction: Bragg’s law
• Scattered X-rays reinforce each other only
when Bragg’s law holds:
Bragg’s law: 2dhkl sin q = nl
17 Jan 2006
10
Structure-Function Analysis
Phase Problem
• If phases hkl and structure factor F(hkl) known:

1
 ( x, y, z )   F (hkl) exp{2i(hx  ky  lz )}
V hkl

1
  F (hkl) expi hkl exp{2i(hx  ky  lz )}
V hkl
• compute the electron density (x,y,z)
– In the electron density build the atomic 3D model
• However, the phases hkl are unknown !
17 Jan 2006
11
Structure-Function Analysis
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
FK, K
FK, H
FH, H
FH, K
• Combine amplitudes FH
with phase K and
inverse-fourier transform
(Taken from: Randy J. Read)
17 Jan 2006
12
Structure-Function Analysis
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
17 Jan 2006
13
Structure-Function Analysis
Building a protein model
• Find structural elements:
– -helices, -strands
• Fit amino-acid sequence
17 Jan 2006
14
Structure-Function Analysis
Building a protein model
• Find structural elements:
– -helices, -strands
• Fit amino-acid sequence
17 Jan 2006
15
Structure-Function Analysis
Effects of resolution on electron density
d=4Å
Note: map calculated with perfect phases
17 Jan 2006
16
Structure-Function Analysis
Effects of resolution on electron density
d=3Å
Note: map calculated with perfect phases
17 Jan 2006
17
Structure-Function Analysis
Effects of resolution on electron density
d=2Å
Note: map calculated with perfect phases
17 Jan 2006
18
Structure-Function Analysis
Effects of resolution on electron density
d=1Å
Note: map calculated with perfect phases
17 Jan 2006
19
Structure-Function Analysis
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
17 Jan 2006
20
Structure-Function Analysis
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
17 Jan 2006
21
Structure-Function Analysis
DNA/Protein structure-function
analysis and prediction
• Protein Structure Determination:
17 Jan 2006
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
22
Structure-Function Analysis
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
17 Jan 2006
23
Structure-Function Analysis
Electron diffraction from 2D crystals:
Nicotinic Acetylcholine Receptor
G-protein coupled receptors
Unwin et al, 2005
17 Jan 2006
24
Structure-Function Analysis
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
17 Jan 2006
25
Structure-Function Analysis
Single particle Electron Microscopy
17 Jan 2006
Select particles
Sort into classes
Average
Reconstruct 3D image
26
Structure-Function Analysis
Cryo EM reconstruction:
Tail of bacteriophage T4
17 Jan 2006
Leiman et al. Cell. 2004 Aug 20;118(4):419-29
27
Structure-Function Analysis
DNA/Protein structure-function
analysis and prediction
• Protein Structure Determination:
17 Jan 2006
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
28
Structure-Function Analysis
1D NMR spectrum of
hen lysozyme (129 residues)
• Too much
overlap in 1D:
 2D
• 1H-1H
• 1H-15N
• 1H-13C
17 Jan 2006
29
Structure-Function Analysis
Step 1: Identification of
amino acid spin systems
R
O
H
C
C
N
C
H
O
H
O
N
C
C
C
H
Cg
N
H
H
H
Amino acid spin system
Amino acid spin system
17 Jan 2006
30
Structure-Function Analysis
2D COSY spectrum of peptide in D2O
Val g
Val 
Thr g
Thr 
17 Jan 2006
31
Structure-Function Analysis
Step 2: Sequential assignment
17 Jan 2006
H(i)-NH(i+1) NOE
R
H
H
f C
N
H
NH(i)-NH(i+1)
NOE
N
C
C
C
O
NH(i)-NH(i+1)
NOE
C
H
O
Cg
N
H
H
H
H(i)-NH(i+1) NOE
32
Structure-Function Analysis
2D NOESY spectrum
Gly
Val
Gly
Leu
Ser
Thr
Phe
Asp
Asn
Asp
• Peptide sequence (N-terminal NH not observed)
• Arg-Gly-Asp-Val-Asn-Ser-Leu-Phe-Asp-Thr-Gly
17 Jan 2006
33
Structure-Function Analysis
Nitroreductase Dimer: 217 residues
• Too much
overlap in 2D:
 3D
• 1H-1H-15N
• 1H-13C-15N
17 Jan 2006
34
Structure-Function Analysis
Structural information from NMR: NOEs
• For macromolecules such as proteins:
17 Jan 2006
– Initial build up of NOE intensity  1/r6
– Between protons that are < 5Å apart
35
Structure-Function Analysis
NOEs in -helices
•
•
•
NH-NH(i,i+1)
H-NH(i,i+3)
H-H(i,i+3)
2.8Å
3.4Å
2.5-4.4Å
O
H
N
H
O
R
• Cytochrome c552 3D 1H-15N NOESY
– H-NH(i,i+3)
– resides 38-47
– form -helix
H
H
R
N
H
N
O
O
H
N
OH
R
R
H
N
R
H
N
O
H
H
H
O
R
N
N
R
R
H O
H
H
17 Jan 2006
O
H
H
R
36
Structure-Function Analysis
NOEs in -strands
•
•
H-NH(i,j)
H-NH(i,j)
H
N
O
R
H
H
N
O
R
H
H
R
O
N
H
H
N
O
17 Jan 2006
2.3Å
3.2Å
H
N
H
R
O
R
H
N
O
R
H
H
R
O
H
H
H
N
O
H
N
H
O
N
R
37
Structure-Function Analysis
Long(er) range NOEs
• Provide information about
– Packing of amino acid side chains
– Fold of the protein
• NOEs observed for
CH of Trp 28 in hen lysozyme
17 Jan 2006
38
• Fine structure in COSY cross peaks
• For proteins 3J(HN.H) useful:
– Probes main chain f torsion angle
R
O
C
f
H
C
N
C
H
O
H
O
N
C
f
C
C
H
• Hen lysozyme:
Cg
N
H
H
H
10
9
Coupling constant (Hz)
Structure-Function Analysis
Spin-spin coupling constants
8
7
6
5
4
3
2
0
20
40
60
80
100
120
140
Residue number
17 Jan 2006
39
Structure-Function Analysis
Structural information from NMR
3. 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
17 Jan 2006
After 68 hours
1H-15N
HSQC spectra for SPH15 in D2O
40
Structure-Function Analysis
Structural information from NMR
• NOEs
– 1H - 2H
– 2H - 40HN
– 3He - 88Hg2
– 3Hd - 55H
1.8-2.5Å
1.8-5.0Å
1.8-5.0Å
1.8-3.5Å
(strong)
(weak)
(weak)
(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
– 11HN-7CO
1.3-2.3Å
17 Jan 2006
10N-6CO
11N-7CO
2.3-3.3Å
2.3-3.3Å
41
• 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
1.2 104
1 10
4
8000
Total energy
Structure-Function Analysis
NMR structure determination:
hen lysozyme
6000
4000
2000
0
10
20
30
40
50
60
70
Structure number
17 Jan 2006
42
Structure-Function Analysis
Solution Structure Ensemble
• Disorder in NMR ensemble
– lack of data ?
17 Jan 2006
– or protein dynamics ?
43
Structure-Function Analysis
DNA/Protein structure-function
analysis and prediction
• Protein Structure Determination:
17 Jan 2006
– X-ray Diffraction
– Electron Microscopy/Diffraction
– NMR Spectroscopy
– Other Spectroscopic methods
44
Structure-Function Analysis
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
17 Jan 2006
45
2
mOD
Structure-Function Analysis
Excited state difference spectra
of Chl and Pheo
keto
0
Chl*/Chl in THF
Pheo*/Pheo in THF
-2
1750
ester
1700
1650
1600
Wavenumber (cm-1)
Keto and ester C=O in Chlorophyll a,
Pheophytin a are redox and environmental probes
17 Jan 2006
46
Structure-Function Analysis
Why is vibrational spectroscopy
sensitive to structure?
17 Jan 2006
ω2
ω2’
X–C–O–H
ω1 O
O–X
ω1’
47
mOD
mOD
Analysis
Structure-Function
H-bond response during the photocycle
weaker stronger
0.5
2 ps, excited state
700 ps, I0
inf, I1
NH
0.0
Tyr42
O
O
-0.5
Arg52
NH2
H
H
Thr50
-1.0
1780
1760
1740
1720
1700
O
O
O
1.5 ps, excited state
800 ps, I0
inf, I1
broken
1
O HN
H
1680
Wavenumber (cm-1)
2
H2N
+
S
Cys69
Glu46
Replace Glu with Gln which
donates weaker H-bond
0
-1
1740
1720
1700
1680
1660
1640
Wavenumber (cm-1)
17 Jan 2006
48
Structure-Function Analysis
Summary
Method
Pro
High resolution
X-ray
No size limits
crystallography
Easy addition of ligands
Crystals required
Phase problem
Electron
microscopy /
diffraction
Single particles possible
Labour intensive
Well suited to
High resolution difficult
membrane proteins
NMR
spectroscopy
In solution
Dynamic information
possible
Resolution variable
Limited size
(< ~30kDa)
(Very) high time
resolution (ps!)
Very sensitive
Technically extremely
complex
Limited applicability
Limited information
Other
spectroscopy
17 Jan 2006
Con
49