Principles of circular dichroism (CD)
and its applications to proteins
José María Delfino
delfino@qb.ffyb.uba.ar
Universidad Nacional de General San Martín 2010
1
2
The context where CD
becomes a useful tool
in biochemistry
3
The folding reaction
N↔U
In general, N = function, U = loss of function
The binding reaction
N + L ↔ NL
Substrate binding to enzymes
Ligand binding to receptors, channels, pumps
Drug binding to target proteins
4
The folding reaction
N↔U
Its importance:
- In general, only the native state N is associated to function
- The polypeptide chain in solution spontaneously adopts the unique
‘fold’ characteristic of the native state N (except where chaperones are
needed)
-An impairment in protein folding may lead to medical disorders:
- In humans: amiloydosis: Alzheimer, Creutzfeldt-Jakob, GerstmannStraussler-Scheinker (GSS), Kuru, emphysema, cystic fibrosis (CF),
Parkinson, cancers, falciform anemia, cataracts, familial
hypercholesterolemia, etc.
- In animals: prion diseases: bovine spongiform encephalopathy (BSE =
mad cow disease), scrapie in ovines, etc.
5
The fundamental experiment of
Christian Anfinsen
(1916-1995, Nobel Prize in Chemistry 1972)
6
The folding reaction N ↔ U
Practical implications:
- To be able to design amino acid sequences that fold in a particular
way to fulfill a specific function,
Please note that the technology to construct and express any protein has been around
for many years (in bacteria, eukaryotic cells and by chemical synthesis):
- Meeting this goal would be of incredible usefulness in medicine and
biotechnology:
↓
- e.g. to design artificial enzymes:
↓
new fermentation processes,
(re)engineering of metabolic paths,
drug production, new materials,
correction of genetic defects
According to many specialists, this is perhaps the most important
problem still unresolved in biochemistry
7
The direct N ↔ U problem:
How does a given amino acid chain
fold?
In general, a sequence folds into a unique 3D structure (‘fold’)
The inverse N ↔ U problem:
How many amino acid sequences
fold into a given ‘fold’?
Many different sequences (even only 5-10% identical) can fold
into a unique ‘fold’, i.e. the folding code is highly degenerate →
there are millions of sequences in nature, but maybe only as
few as ~2000 ‘folds’
8
MTEMKDDFAKLEEQFDAKLGIFALDTGTNR
TVAYRPDERFAFASTIKALTVGVLLQQKSI
EDLNQRITYTRDDLVNYNPITEKHVDTGMT
LKELADASLRYSDNAAQNLILKQIGGPESL
KKELRKIGDEVTNPERFEPELNEVNPGETQ
DTSTARALVTSLRAFALEDKLPSEKRELLI
DWMKRNTTGDALIRAGVPDGWEVADKTGAA
SYGTRNDIAIIWPPKGDPVVLAVLSSRDKK
DAKYDDKLIAEATKVVMKALNMNGDKLPSE
?
9
MTEMKDDFAKLEEQFDAKLGIFALDTGTNR
TVAYRPDERFAFASTIKALTVGVLLQQKSI
EDLNQRITYTRDDLVNYNPITEKHVDTGMT
LKELADASLRYSDNAAQNLILKQIGGPESL
KKELRKIGDEVTNPERFEPELNEVNPGETQ
DTSTARALVTSLRAFALEDKLPSEKRELLI
DWMKRNTTGDALIRAGVPDGWEVADKTGAA
SYGTRNDIAIIWPPKGDPVVLAVLSSRDKK
DAKYDDKLIAEATKVVMKALNMNGDKLPSE
β-lactamase
10
The sequence databases:
www.ncbi.nlm.nih.gov National Center for Biotechnology Information
www.expasy.ch SwissProt
pir.georgetown.edu Protein Information Resource
www.srs.ebi.ac.uk Sequence Retrieval System
www.uniprot.org The UniProt effort to unify sequence databases
The non-redundant (nr) database includes ~10.8 million
sequences, representing ~3.7 thousand million “letters” (amino
acids) (April 2010)
The 3D structure database:
Protein Data Bank: www.rcsb.org
This database of atomic coordinates of known 3D structures of
proteins, nucleic acids, complexes and other macromolecules
includes ~ 65000 structures, determined by X-ray
crystallography, NMR and electron microscopy (April 2010)
11
Sequence of IFABP (in FASTA format):
>2IFB:_|PDBID|CHAIN|SEQUENCE
AFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNKFTVKESSNFRNI
DVVFELGVDFAYSLADGTELTGTWTMEGNKLVGKFKRVDNGKELIAVREISGNELIQT
YTYEGVEAKRIFKKE
Atomic coordinates of IFABP (in pdb format):
HEADER
COMPND
COMPND
SOURCE
AUTHOR
REVDAT
REVDAT
JRNL
JRNL
JRNL
JRNL
JRNL
JRNL
JRNL
REMARK
REMARK
...
REMARK
REMARK
SEQRES
SEQRES
...
FORMUL
FORMUL
HELIX
HELIX
SHEET
SHEET
...
FATTY ACID-BINDING PROTEIN
05-DEC-90
2IFB
INTESTINAL FATTY ACID BINDING PROTEIN (HOLO FORM)
2 (/I-FABP$)
RAT (RATTUS $RATTUS) EXPRESSED IN (ESCHERICHIA $COLI)
J.C.SACCHETTINI,J.I.GORDON,L.J.BANASZAK
2
30-APR-94 2IFBA
3
HETATM CONECT
1
15-JAN-92 2IFB
0
AUTH
J.C.SACCHETTINI,J.I.GORDON,L.J.BANASZAK
TITL
CRYSTAL STRUCTURE OF RAT INTESTINAL
TITL 2 FATTY-ACID-BINDING PROTEIN. REFINEMENT AND ANALYSIS
TITL 3 OF THE ESCHERICHIA $COLI-DRIVED PROTEIN WITH BOUND
TITL 4 PALMITATE
REF
J.MOL.BIOL.
V. 208
327 1989
REFN
ASTM JMOBAK UK ISSN 0022-2836
070
1
2
2IFB
2IFB
2IFB
2IFB
2IFB
2IFBA
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
4 CORRECTION. REVISE ATOM NAMING AND ORDERING FOR HET GROUP
4 PLM TO FOLLOW PDB SPECIFICATIONS. 30-APR-94.
1
131 ALA PHE ASP GLY THR TRP LYS VAL ASP ARG ASN GLU ASN
2
131 TYR GLU LYS PHE MET GLU LYS MET GLY ILE ASN VAL VAL
2IFBA 3
2IFBA 4
2IFB 26
2IFB 27
2
3
1
2
1
2
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
PLM
C16 H32 O2
HOH
*61(H2 O1)
A1 ASN
13 MET
A2 ASN
24 HIS
B1 6 ASP
3 GLU
B1 6 ASP
34 GLU
21 1
33 1
12 0
43 -1
N
ILE
40
O
GLY
4
2
3
4
5
6
1
7
8
9
10
11
12
13
14
15
16
IFABP
(intestinal fatty acid
binding protein)
38
39
40
41
42
43
and it continues…
12
(pdb file continues)
CRYST1
ORIGX1
ORIGX2
ORIGX3
SCALE1
SCALE2
SCALE3
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
...
MASTER
END
36.800
56.900
31.900 90.00
0.027174 0.000000 0.012099
0.000000 0.017575 0.000000
0.000000 0.000000 0.034315
0.027174 0.000000 0.012099
0.000000 0.017575 0.000000
0.000000 0.000000 0.034315
1 N
ALA
1
5.210
2 CA ALA
1
4.880
3 C
ALA
1
6.063
4 O
ALA
1
5.895
5 CB ALA
1
4.579
6 N
PHE
2
7.269
7 CA PHE
2
8.399
8 C
PHE
2
9.117
9 O
PHE
2
10.100
14
0
1
2
11
0
114.00
6.162
7.329
8.279
9.480
6.942
7.755
8.620
9.093
9.827
0
90.00 P 21
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
2.340 1.00
3.147 1.00
3.211 1.00
3.380 1.00
4.593 1.00
3.072 1.00
3.319 1.00
2.072 1.00
2.119 1.00
6 1136
1
2
63.97
54.91
45.04
44.89
52.86
30.47
22.66
24.99
26.08
28
11
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
IFABP
2IFBA 43
2IFB1226
The atomic coordinates (in Å) are highlighted in the box
13
The degrees of freedom of the polypeptide chain:
The main chain (‘backbone’) and the torsion angles phi and psi
In general, the omega angle (peptide bond torsion) adopts values close to 180º and,
rarely, close to 0o → both consistent with planarity
e.g. the peptide Ala-Ala-Ala
14
The polypeptide chain as a succession of planes
connected by the vertices and related through
the torsions phi and psi
15
The Ramachandran plot
In a folded polypeptide chain, the phi/psi pairs adopt characteristic values
that define the different types of secondary structure: alpha, beta and others
16
What does it mean to know the conformation of a
protein?
To define the set of torsion angles for each amino acid along the
polypeptide chain,
i.e., to know phi/psi/omega and all chi (chi1, chi2, etc. of the side
chains) becomes equivalent
↓
to know the atomic coordinates (x,y,z)
of each atom,
i.e. the information deposited in the PDB bank (www.rcsb.org)
Exercise: extract a structure from the PDB bank and represent it graphically
with the aid of a visualization program (such as RasMol, PyMol, VMD, or
SwissPDBViewer), measure some characteristic torsion angles
17
The Levinthal’s paradox
In 1969 Cyrus Levinthal theoretically addressed the fact
that if the search for the folded form was random, the
polypeptide chain would have to sample an astronomical
number of conformations from the unfolded ensemble U
to reach the native state N.
Such search would take more time than the age of the
universe! Therefore, this process would be incredibly
unlikely.
As an example:
• Suppose only 2 possible conformations for each amino acid
• Then, a polypeptide of 100 amino acid residues would adopt 2100 (=1.27.
1030) possible conformations
• If each conformation would turn into the next in 1 ps (1 picosecond = 10-12
s), then the time required to sample the whole conformational space to find
the native state would amount to ~1018 seconds or ~1010 years!
However, proteins fold in miliseconds to seconds!
18
Not all possible conformations are
sampled, there are ‘paths’ in the
‘folding landscape’ to reach the
native state N
↓
The energy surface is not like a
golf field!
19
The new view:
energy funnels
(P Wolynes)
20
The shape of energy funnels (K Dill)
21
Experimental techniques to study
the folding reaction (a non-exhaustive list)
Spectroscopies
- Circular dichroism (CD)
- Fluorescence: intrinsic (Trp), probes (e.g. ANS)
- UV absorption
- Nuclear magnetic resonance (NMR)
Size and shape determination
- Light scattering (DLS)
- X ray dispersion (e.g. SAXS)
- Size exclusion chromatography (e.g. SEC-FPLC)
Chemical alteration
- Limited proteolysis
- Chemical modification
- H/D amide exchange
Variant construction
- Site-directed mutagenesis
- Peptide synthesis
- Expression of variants (e.g. truncated, circularly permuted)
- Fragment complementation
Thermodynamics
Functional studies
- Isothermal titration calorimetry (ITC)
- Differential scanning calorimetry (DSC)
- Enzymatic catalysis
- Ligand binding
22
Highlights on key conformational techniques:
Size Exclusion Chromatography (SEC) combined with Light Scattering (LS)
&
Chemical Cross-linking (e.g. with a bifunctional reagents such as DSS):
- aggregation state of the protein, overall shape and volume
Circular Dichroism:
- far UV region: secondary (and tertiary) structure
- near UV region: tertiary structure
- ligand-induced bands: features of the binding site
Fluorescence Emission:
- Trp environment
- Quenching effects: map accessibility of the core region
- Interaction between a ligand and a fluorophore:
Measurement of the affinity for ligands
23
Highlights on key conformational techniques:
Size Exclusion Chromatography (SEC) combined with Light Scattering (LS)
&
Chemical Cross-linking (e.g. with a bifunctional reagents such as DSS):
- aggregation state of the protein, overall shape and volume
Circular Dichroism:
- far UV region: secondary (and tertiary) structure
- near UV region: tertiary structure
- ligand-induced bands: features of the binding site
Fluorescence Emission:
- Trp environment
- Quenching effects: map accessibility of the core region
- Interaction between a ligand and a fluorophore:
Measurement of the affinity for ligands
24
25
An intuitive approach
to understand the nature
of polarized light and
its interaction with matter
26
Unpolarized, linearly (or plane)
polarized, and circularly polarized light
27
What is
optical
rotatory
dispersion
(ORD)?
α=[α]cl
nL≠nR
α (Ó φ)
28
Two systems to represent a light beam
y
y
z
x
x
29
Three simple exercises to intuitively
understand the (general) nature of
polarized light and the CD phenomenon
1. A plane polarized light beam results from the sum of two in phase
circularly polarized light beams of opposite sign (R and L)
What would happen if the constituent beams were out of phase?
2. A circularly polarized light beam results from the sum of two perpendicular
plane polarized light beams dephased +¼ wavelength (= +π/2)
What would happen if the dephasing were instead -¼ wavelength (= -π/2)?
What would be the outcome if they were in phase (= 0)?
Remember this point to understand the function of the Pockels cell (see block diagram of the apparatus)!
3. A plane polarized light beam -of which one of the circular components (R
or L) were differentially absorbed (by a dichroic sample)- would result in an
elliptically polarized light beam
What would be the orientation of the major axis of the ellipse?
What would the result be if -in addition to the differential absorption- dephasing would also occur?
30
Electromagnetic waves and circular dichroism:
an animated tutorial
By András Szilágyi (szia@enzim.hu)
www.enzim.hu/~szia/cddemo/edemo0.htm
31
Circular Dichroism (CD), a pictorial view
32
What is Circular Dichroism (CD)?
CD is the differential absorption -by an asymmetric
chromophoric molecule (the polypeptide chain in our
case)- of right and left circularly polarized light beams.
The magnitude of CD is measured by the ellipticity (Ө,
theta), an angle parameter expressed in (mili)degree
units.
33
What is Circular
Dichroism (CD)?
Two equivalent
expressions:
AL≠AR
θ=[θ]cl
ΔA=Δεcl
34
Plane polarized
light turns into
elliptically
polarized light
by the
differential
absorption of
an optically
active
chromophore
35
However, both ORD and CD are different outcomes of
the same physical phenomenon, i.e. the interaction of
polarized light (ER and EL) with chiral molecules
In ORD, the detection consists in evaluating the change
in the velocity of the beams (by measuring the change in
the index of refraction nR ≠ nL)
In CD, the detection consists in evaluating the change in
the amplitudes: |ER| and |EL| of the beams (through the
change in absorption: εR ≠ ε L)
36
If CD and ORD are indeed so intimately related,
the information derived from each technique is
redundant
In fact, each spectrum can be converted to the
other via the Kronig-Kramers transforms:
37
Nowadays CD is used more often than ORD
Superior CD instrumentation (alternate nature of the
detection by CD)
Band shapes in CD are more narrow and of a single
sign, leading to less spread, thus achieving better
spectral resolution and facilitating the assignment
The asymmetry of chromophores in proteins (amides,
aromatic groups and disulfide bridges) is induced by
their interaction with neighboring groups (the chemical
environment)
Estimate secondary structure content
Uses
of CD
Detect conformational changes
Measure ligand binding
38
The ORD spectrum looks like the derivative (but it
is not) of the CD spectrum, however, the
dependence with λ is different
For this reason, it is possible to measure optical activity
in regions far from the absorption maximum (e.g. in sugars)
By contrast, the high UV absorption of proteins allows
the measurement of CD, the concentration is expressed
in terms of the mean amino acid residue weight (MRW):
MRW = MW / #res
39
Physical conditions allowing the existence of optical activity
Optically
active
transition
40
The Cotton effect is the
outcome of the
phenomenon of
interaction of polarized
light with chiral matter
Transitions as seen by
ORD (dispersive,
dashed lines) or
CD (absorptive, solid
lines):
41
The (quasi)linear relationship existing between
molar ellipticity ([θ]) and the difference in the molar
extinction coefficients (Δε)
Differential LambertBeer’s law
Definition of
absorbance A
42
Molar ellipticity ([θ]) and the difference in the
molar extinction coefficients (Δε) are equivalent
measurements (convertible by a constant factor)
43
How come [θ] = 3300 Δε?
44
The basic equations of ORD and CD
ORD
CD
The units:
ORD
O
cm2 dmol-1
CD
O
cm2 dmol-1
45
The CD instrument: the spectropolarimeter
•Compact benchtop design
•Air cooled 150W Xenon lamp or Water cooled 450W Xenon lamp
•Highest Signal-to-Noise ratio. Range of precise temperature control
accessories
Automated titration and stopped-flow accessories
•Spectra Manager™ II software for control and data analysis
•Spectra Manager™ CFR option for 21 CFR 11 compliance
•Flexible design allows field upgrades for different measurement modes
and accessories as applications evolve.
Measurement modes and Hyphenated techniques
Standard
•Circular Dichroism/UV/VIS absorbance
Optional
•Linear Dichroism (LD)
•Optical Rotatory Dispersion (ORD)
•Total Fluorescence (TF)
•Scanning EM Fluorescence
•Fluorescence Detected CD (FDCD)
•Stopped-Flow CD
•Stopped-Flow Absorbance
•Stopped-Flow Fluorescence
•Chiral HPLC Detection
•Magnetic CD (MCD)
•Near Infrared CD (NIRCD)
Optional Accessories
•Peltier cell holders, single and six position
•Scanning emission monochomator
•Automatic titration system
•2, 3, and 4 syringe stopped-flow systems
•LD, ORD attachments
•Permanent, electro and super-conducting magnets
•Near IR extended detection
•And many more!
J-815 Circular Dichroism Spectrometer
Optional Program
•Protein secondary structure estimation program
•Detatured protein analysis program
•Multi-WL variable temperature measurement program
•Macro command program
•And many more!
46
The CD instrument: the spectropolarimeter
Pockels cell
Calibration:
CSA
Δε -4.9 @ 192.5 nm
Δε +2.36 @ 290.5 nm
47
The innards of the CD instrument:
A block diagram
Pockels cell
Radiation is split into the two circularly
polarized components by passage
through a modulator (usually a
piezoelectric crystal such as quartz)
subjected to an alternating (50 kHz)
electric field. The modulator will transmit
each of the two components in turn.
If, after passage through the sample, the components are not absorbed (or are absorbed to
the same extent), combination of the components would regenerate radiation polarized in
the original plane. However, if one of the components is absorbed by the sample to a greater
extent than the other, the resultant (combined component) radiation would now be
elliptically polarized, i.e., the resultant would trace out an ellipse.
48
Practical aspects I:
Manufacturers: Horiba-Jobin Yvon, Jasco, AVIV
More potent light sources vs. efficient
ΔA ~ 10-4 A
photodetectors (PMT), enhanced electronics to suppress noise
1 to 10 cm cells in the near UV region: to detect weak signals, and
1, 0.5, 0.1 mm (and even 0.05 and 0.01 mm!) cells in the far UV region,
to minimize solvent absorption
Continuous N2 flow: to avoid ozone damage to the optics (mirrors)
It is essential to accurately know the protein concentration in the
sample: by spectrophotometry (using a reliable ε value), or by
quantitative amino acid analysis
49
Practical aspects II:
Reduce spectral noise via:
- sum of several scans/digital smoothing (Savitzky-Golay, FT)
- increase data collection time (especially so in the very far UV
region, where the absorption is high, e.g. 1 nm/min and 4 sec time constant).
In general, follow the rule of thumb:
Scan speed (nm/sec) times Time constant (sec) < 0.33
- alternate spectrum collection of the sample with blanks (buffer)
and standards (known protein samples, etc.)
Keep transparency of buffers (choice of phosphates, perchlorate,
borates,Tris, in this order) and additives (DTT or βME < 1 mM, EDTA < 0.1
mM)
CD measurements can be carried out on samples that disperse light
significantly (e.g. membrane proteins in micelles or liposomes). MOPS,
lubrol and SDS are acceptable
The information content of the spectrum increases a lot at low wavelengths
(if possible, scan up to λ < 190 nm)
50
51
How CD
becomes useful to
understand protein structure
52
Nowadays, CD is used more often than ORD
Availability of superior instrumentation (alternate
nature of detection in CD)
Less ‘spread’ of bands in CD -of only one-sign and
more narrow- allows better spectral resolution and
easier assignment
Chromophore asymmetry in proteins (amide groups,
aromatic groups and disulfide bridges) is induced by the
chirality of the chemical environment
Estimate the secondary structure content of a protein
Main
uses
Detect conformational changes
Measure ligand binding
53
Common applications of circular dichroism
(CD) in proteins and peptides:
- Estimate secondary structure content
- Evaluate conformational changes
- Measure ligand binding phenomena
The possibility exists to carry out both
equilibrium and kinetic experiments
54
The electronic transitions in proteins:
The peptide bond: n→π* (br, w) ~ 210 nm
π→ π* (sh, s) ~ 190 nm
Cystine:
S
χ3
S
Far UV region
(180-250 nm)
…and the aromatic residues (see below)
Aromatic residues (optically inactive per se, but
placed in asymmetric environments):
W, Y, F, H,
Cystine (w, ~ 280 nm)
Near UV region
(250-340 nm)
… also prosthetic groups (e.g. heme) and
metalloproteins
55
Circular Dichroism (CD) (CD)
CD in the far UV region (180-240 nm) -where the
peptide bond absorbs light- reports on the overall
content of secondary structure
56
Circular Dichroism (CD)
50
ES-βL
S126C S265C ES-βL
S126C ES-βL
S265C ES-βL
0
0 M, WT
42
0.0 M, trunc.
2.0 M, trunc.
5.0 M, trunc.
28
6.6 M, trunc.
6.6 M, WT
-50
14
-100
0
-150
-14
250
260
270
280
290
300
310
Wavelength (nm)
320
330
250
260
270
280
290
300
310
320
Wavelength (nm)
Javier Santos
The CD in the near UV region (240-340 nm) -where the
side-chain chromophores of W,Y,F,H and the disulfide
bonds absorb light- reveal features of the tertiary structure
(asymmetric environments): a ‘fingerprint’ of the protein
57
Estimate secondary structure content
The reference
spectra (basis
set) for the
different types of
secondary
structure: α
helix, β sheet,
and random coil
A critical point is the
wise choice of
standards
Based on amino acid
polymers: poly-K, poly-E
(Fasman)
Problem: dependency on
the length of helices,
sheets or coils, uncertain
contribution of turns
Based on known 3D
structures taken from
the PDB: α helix,
parallel and antiparallel β sheet, type I,
II and III β turns
(Wetlaufer)
58
Spectral deconvolution into standard components
59
There are several methods to deconvolute (decompose) spectra, so that
secondary structure content can be extracted:
SSE
CONTIN
BELOK
VARSLC 1
Self-consistent
LINCOMB/CCA (convex constraint analysis)
BPNN (use of neural networks)
SOM-BPN
PROT CD
Check the DICHROWEB site: www.cryst.bbk.ac.uk/cdweb/html
Nevertheless, problems persist in regard to the reliability of the basis sets (e.g.
there is less information on β structure than on α structure), and the variable
contribution of aromatic residues in this spectral region (see below)
60
All α proteins
61
All
β proteins
62
α + β proteins
63
α/β proteins
64
Disordered proteins
65
The contribution of aromatic residues
66
A cautionary note whenever interpreting the
contributions to [θ]222!
67
68
Ligand binding: calcium binding to calmodulin
69
The binding of an intercalator molecule to dsDNA
70
Two coupled equilibria: the folding of protein
P and the binding of anionic ligands
71
Two structurally related proteins exhibiting
very different folding mechanisms:
bovine alpha-lactalbumin (α-LA) and lysozyme (HEWL)
HEWL
apo α-LA
HEWL
apo α-LA
72
CD reveals the
presence of
folding
intermediates:
α-LA vs. HEWL
U
U
N
N
HEWL
MG α-LA
(Kuwajima)
The ‘molten
globule’ (MG) state
of α-LA conserves
the dichroic signal
in the far UV zone,
but loses the
signal in the near
UV region
U/MG
U
N
N
HEWL
α-LA
73
Another example of a molten globule (MG):
Conservation of secondary structure with loss of
tertiary interactions, a critical step for the insertion
of colicin A in membranes
pH 2
pH 2
pH 7
pH 7
74
The channel polypeptide
P190
changes its
conformation as a
function of pH
75
Folding kinetics detected
by CD
(time resolved CD)
The case of cytochrome c
(Elöve, Englander, Roder)
76
Folding kinetics
of
HEWL and α-LA
(Kuwajima)
77
78
79
80
81
82
83
84
Some sites of interest on circular dichroism (CD):
Brief introduction, tutorial with examples and programs: www.imbjena.de/ImgLibDoc/cd/index.htm
Brief critical analysis of the technique:
www.cryst.bbk.ac.uk/PPS2/course/section8/ss_960531_21.html
CD class with applications to proteins and nucleic acids:
www.newark.rutgers.edu/chemistry/grad/chem585/lecture1.html
Practical aspects of conformational transitions:
www.ap-lab.com/circular_dichroism.htm
Basic concepts and instrumentation: www.ruppweb.org/cd/cdtutorial.htm
Animations on polarized light: www.enzim.hu/~szia/cddemo/edemo0.htm
A database on CD spectra (under construction): pcddb.cryst.bbk.ac.uk
On the deconvolution of CD spectra with DICHROWEB:
www.cryst.bbk.ac.uk/cdweb/html
Simple tutorial with a focus on applications: wwwstructure.llnl.gov/cd/cdtutorial.htm
85
Reference books
1979
2005
1997
1996
Para ver esta película, debe
disponer de QuickTime™ y de
un descompresor .
2009
1984
1998
1980
86
The Greenfield papers:
Norma J Greenfield
‘Determination of the folding of proteins as a function of denaturants, osmolytes or
ligands using circular dichroism’
Nat Protoc. 2006 ; 1(6): 2733-2741
Norma J Greenfield
'Using circular dichroism collected as a function of temperature to determine the
thermodynamics of protein unfolding and binding interactions’
Nat Protoc. 2006 ; 1(6): 2527-2535
Norma J Greenfield
‘Using circular dichroism spectra to estimate protein secondary structure’
Nat Protoc. 2006 ; 1(6): 2876-2890
Norma J Greenfield
‘Analysis of the kinetics of folding of proteins and peptides using circular dichroism’
Nat Protoc. 2006 ; 1(6): 2891-2899
87
88
El diagrama de Ramachandran II
¿Por qué los pares phi/psi no pueden adoptar cualquier valor?
Impedimento estérico que involucra la cadena principal y las cadenas laterales
89
El diagrama de Ramachandran III
Los puntos azules representan pares de ángulos phi/psi medidos en una
proteína real extraída del Protein Data Bank (www.rcsb.org)
90
El diagrama de
Ramachandran IV
Los aminoácidos Gly y Pro
adoptan valores de phi/psi
atípicos:
Gly
(sin cadena lateral)
→ mayor flexibilidad
valores phi/psi especiales
Pro
(cadena lateral ciclada al N)
→ valor de phi fijo
sólo varía psi
91
Las cadenas laterales de los aminoácidos también adoptan
conformaciones características → los ángulos de torsión chi
(chi1, chi2, etc.)
92
Los ángulos de torsión chi adoptan valores característicos
de acuerdo con el tipo de aminoácido (p.ej. Leu)
→ existen bibliotecas de rotámeros
93
The Cotton effect is
the manifestation of
the interaction
phenomenon of
polarized light with
the chiral matter
Here it is how it
looks like by ORD
and CD:
94
El instrumento de medida:
el espectropolarímetro
Celda de Pockels
95
La calibración del
espectropolarímetro
CSA
Δε -4.9 @ 192.5 nm
Δε +2.36 @ 290.5 nm
Rango A280 ~ 0.4-1.0 en proteína
96