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Transcript
Principles of circular dichroism (CD)
and its applications to proteins
José María Delfino
[email protected]
Departamento de Química Biológica e
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB, UBA-CONICET)
Facultad de Farmacia y Bioquímica
Universidad de Buenos Aires
Junín 956, 1113 Buenos Aires, Argentina
UNSAM, abril 2012
La reacción de plegamiento
NU
Importancia del problema:
- En general, sólo el estado plegado N está asociado a la función
- En general, la cadena polipeptídica en solución adopta
espontáneamente el plegamiento característico del estado nativo N
(salvo en casos particulares en que son necesarias chaperonas)
- Deficiencias en el proceso de plegamiento conducen a enfermedades:
- En humanos: amiloidosis: Alzheimer, Creutzfeldt-Jakob,
Gerstmann-Straussler-Scheinker (GSS), Kuru, enfisema,
fibrosis quística, Parkinson, algunos cánceres,
anemia falciforme, cataratas, hipercolesterolemia familiar, etc.
- En animales: enfermedades causadas por priones:
encefalopatía espongiforme bovina (BSE, vaca loca),
scrapie en ovinos, etc.
La reacción de plegamiento N U
Implicancias prácticas del problema:
- Ser capaz de diseñar secuencias de aminoácidos que se plieguen
de un modo particular para cumplir funciones determinadas,
adviértase aquí que desde hace años existe la tecnología para expresar cualquier
proteína (en bacterias o células eucariotas, y aún por síntesis química):
- La concreción de este objetivo sería de increible utilidad en
medicina y en biotecnología:
- P.ej. fabricación de enzimas artificiales:
nuevos procesos de fermentación,
ingeniería de nuevas vías metabólicas,
producción de fármacos y nuevos materiales,
corrección de defectos genéticos
Según muchos especialistas, quizás éste sea el mayor problema aún
no resuelto en bioquímica
El problema N 㲗 U directo:
¿Cómo se pliega una dada secuencia
de aminoácidos?
En general, una secuencia se pliega para dar una única
estructura 3D (fold)
El problema N 㲗 U inverso:
¿Cuántas secuencias de aminoácidos se pliegan de un modo dado?
Muchas secuencias diferentes (aún similares en sólo 5-10%)
pueden plegarse de un único modo (o fold): el código de
plegamiento está altamente degenerado existen millones de
secuencias en la naturaleza, pero quizás sólo ~2000 folds
Los grados de libertad de la cadena
polipeptídica:
La cadena principal (backbone) y los ángulos de torsión phi y psi
También existe el ángulo omega (torsión de la propia unión peptídica) que adopta en
general valores de 180º y, raramente, de 0o ambos consistentes con la planaridad
Ej. el péptido Ala-Ala-Ala
La cadena polipeptídica como sucesión de planos
conectados por los vértices, relacionados a través de
las torsiones phi y psi
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)
Las cadenas laterales de los aminoácidos también adoptan
conformaciones características los ángulos de torsión chi
(chi1, chi2, etc.)
Los ángulos de torsión chi adoptan valores característicos
de acuerdo con el tipo de aminoácido (p.ej. Leu)
existen bibliotecas de rotámeros
La paradoja de Levinthal
En 1969 Cyrus Levinthal demostró teóricamente que si
la búsqueda de la conformación plegada fuera al azar,
la cadena polipeptídica debería muestrear un número
astronómico de conformaciones desde el estado
desplegado U hasta alcanzar el estado nativo N.
La búsqueda tomaría tanto tiempo (mayor que la edad
del universo!), de modo que el proceso sería
increíblemente improbable.
Esto es:
• Suponiendo sólo 2 conformaciones posibles para cada aminoácido
• Entonces un polipéptido de 100 residuos poseería 2100 (=1.26. 1030)
posibles conformaciones
• Si cada conformación se convirtiera en otra en 1 ps (picosegundo 10-12 s)
Entonces, el tiempo requerido para el proceso de plegamiento sería de
~1018 segundos o ~1010 años!
Sin embargo, las proteínas se pliegan en milisegundos (ms) a minutos
No se muestrean todas las
conformaciones posibles,
sino que existen “caminos” para
alcanzar el estado nativo N
La superficie de energía no es
un campo de golf!
Las bases de datos de secuencias:
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 UniProt unifica las bases de datos de secuencias
La base de secuencias no redundante (nr) incluye ~17.37
millones de secuencias, que representan ~5.96 mil millones de
“letras” (aminoácidos) (28 febrero 2012)
La base de datos de estructuras 3D:
Protein Data Bank: www.rcsb.org
Esta base de coordenadas atómicas de estructuras 3D de
proteínas, ácidos nucleicos, complejos y otras macromoléculas
incluye ~79700 estructuras, determinadas por cristalografía de
rayos X, NMR y microscopía electrónica (28 febrero 2012)
Algunas herramientas en Expasy (I): http://expasy.org/tools/
http://www.isb-sib.ch/
Primary structure analysis
ProtParam - Physico-chemical parameters of a protein sequence (amino-acid and atomic compositions, isoelectric point, extinction coefficient, etc.)
Compute pI/Mw - Compute the theoretical isoelectric point (pI) and molecular weight (Mw) from a UniProt Knowledgebase entry or for a user sequence
ScanSite pI/Mw - Compute the theoretical pI and Mw, and multiple phosphorylation states
MW, pI, Titration curve - Computes pI, composition and allows to see a titration curve
Scratch Protein Predictor
HeliQuest - A web server to screen sequences with specific alpha-helical properties
Radar - De novo repeat detection in protein sequences
REP - Searches a protein sequence for repeats
REPRO - De novo repeat detection in protein sequences
TRUST - De novo repeat detection in protein sequences
XSTREAM - De novo tandem repeat detection and architecture modeling in protein sequences
SAPS - Statistical analysis of protein sequences at EMBnet-CH [Also available at EBI]
Coils - Prediction of coiled coil regions in proteins (Lupas's method) at EMBnet-CH [Also available at PBIL]
Paircoil - Prediction of coiled coil regions in proteins (Berger's method)
Paircoil2 - Prediction of the parallel coiled coil fold from sequence using pairwise residue probabilitis with the Paircoil algorithm.
Multicoil - Prediction of two- and three-stranded coiled coils
2ZIP - Prediction of Leucine Zippers
ePESTfind - Identification of PEST regions
HLA_Bind - Prediction of MHC type I (HLA) peptide binding
PEPVAC - Prediction of supertypic MHC binders
RANKPEP - Prediction of peptide MHC binding
SYFPEITHI - Prediction of MHC type I and II peptide binding
ProtScale - Amino acid scale representation (Hydrophobicity, other conformational parameters, etc.)
Drawhca - Draw an HCA (Hydrophobic Cluster Analysis) plot of a protein sequence
Más herramientas en Expasy (II): http://expasy.org/tools/
http://www.isb-sib.ch/
Secondary structure prediction
AGADIR - An algorithm to predict the helical content of peptides
APSSP - Advanced Protein Secondary Structure Prediction Server
CFSSP - Chou & Fasman Secondary Structure Prediction Server
GOR - Garnier et al, 1996
HNN - Hierarchical Neural Network method (Guermeur, 1997)
HTMSRAP - Helical TransMembrane Segment Rotational Angle Prediction
Jpred - A consensus method for protein secondary structure prediction at University of Dundee
JUFO - Protein secondary structure prediction from sequence (neural network)
NetSurfP - Protein Surface Accessibility and Secondary Structure Predictions
NetTurnP - Prediction of Beta-turn regions in protein sequences
nnPredict - University of California at San Francisco (UCSF)
Porter - University College Dublin
PredictProtein - PHDsec, PHDacc, PHDhtm, PHDtopology, PHDthreader, MaxHom, EvalSec from Columbia University
Prof - Cascaded Multiple Classifiers for Secondary Structure Prediction
PSA - BioMolecular Engineering Research Center (BMERC) / Boston
PSIpred - Various protein structure prediction methods at Bloomsbury Centre for Bioinformatics
SOPMA - Geourjon and Deléage, 1995
Scratch Protein Predictor
DLP-SVM - Domain linker prediction using SVM at Tokyo University of Agriculture and Technology
Tertiary structure
Tertiary structure analysis
iMolTalk - An Interactive Protein Structure Analysis Server (currently down)
MolTalk - A computational environment for structural bioinformatics
COPS - Navigation through fold space and the instantaneous visualization of pairwise structure similarities
PoPMuSiC - Prediction of thermodynamic stability changes upon point mutations; design of modified proteins
Seq2Struct - A web resource for the identification of sequence-structure links
STRAP - A structural alignment program for proteins
TLSMD - TLS (Translation/Libration/Screw) Motion Determination
TopMatch-web - Protein structure comparison
Aún más herramientas en Expasy (III): http://expasy.org/tools/
Tertiary structure prediction
Homology modeling
SWISS-MODEL - An automated knowledge-based protein modelling server
CPHmodels - Automated neural-network based protein modelling server
ESyPred3D - Automated homology modeling program using neural networks
Geno3d - Automatic modelling of protein three-dimensional structure
http://www.isb-sib.ch/
Threading
Phyre (Successor of 3D-PSSM) - Automated 3D model building using profile-profile matching and secondary structure
Fugue - Sequence-structure homology recognition
HHpred - Protein homology detection and structure prediction by HMM-HMM comparison
LOOPP - Sequence to sequence, sequence to structure, and structure to structure alignment
SAM-T08 - HMM-based Protein Structure Prediction
PSIpred - Various protein structure prediction methods (including threading) at Bloomsbury Centre for Bioinformatics
Ab initio
HMMSTR/Rosetta - Prediction of protein structure from sequence
Assessing tertiary structure prediction
Anolea - Atomic Non-Local Environment Assessment
LiveBench - Continuous Benchmarking of Structure Prediction Servers
NQ-Flipper - Validation and correction of asparagine and glutamine side-chain amide rotamers in protein structures solved by X-ray crystallography
PROCHECK - Verification of the stereochemical quality of a protein structure
ProSA-web - Recognition of errors in 3D structures of proteins
QMEAN - Server for Model Quality Estimation
What If - Protein structure analysis program for mutant prediction, structure verification, molecular graphics
Quaternary structure
MakeMultimer - Reconstruction of multimeric molecules present in crystals
EBI PISA - Protein Interfaces, Surfaces and Assemblies
PQS - Protein Quaternary Structure Query form at the EBI
ProtBud - Comparison of asymmetric units and biological units from PDB and PQS
Molecular modeling and visualization tools
Swiss-PdbViewer - A program to display, analyse and superimpose protein 3D structures
SwissDock - Docking of small ligands into protein active sites with EADock DSS
…y la lista sigue …
Las predicciones a partir de la secuencia de aminoácidos
P. ej.
Las predicciones a partir de la secuencia de aminoácidos
P. ej.
¿Qué significa conocer la conformación de una
proteina?
Definir el conjunto de los ángulos de torsión para cada
aminoácido de una proteína,
esto es, phi/psi/omega y todos los chi (chi1, chi2, etc.) resulta
equivalente a
conocer las coordenadas atómicas (x,y,z)
de cada uno de los átomos,
esto es, la información depositada en el banco pdb
(www.rcsb.org)
Ejercicio: extraer una estructura del banco pdb y representarla mediante algún
programa de visualización (Pymol, VMD, RasMol o SwissPDBViewer),
medir algunos ángulos de torsión característicos
Secuencia de IFABP (en formato FASTA):
>2IFB:_|PDBID|CHAIN|SEQUENCE
AFDGTWKVDRNENYEKFMEKMGINVVKRKLGAHDNLKLTITQEGNKFTVKESSNFRNI
DVVFELGVDFAYSLADGTELTGTWTMEGNKLVGKFKRVDNGKELIAVREISGNELIQT
YTYEGVEAKRIFKKE
Coordenadas atómicas de IFABP (en formato PDB):
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 2IF B
0
AUTH
J.C.SACCHETTINI,J.I.GORDON,L.J.BANASZAK
TITL
CRYSTAL STRUCTURE OF RAT INTESTINAL
TITL 2 FATTY -ACID -BINDING P ROTEIN. 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
2
2IFB
3
2IFB
4
2IFB
5
2IFB
6
2IFBA 1
2IFB
7
2IFB
8
2IFB
9
2IFB 10
2IFB 11
2IFB 12
2IFB 13
2IFB 14
2IFB 15
2IFB 16
4 CORRECTION. REVISE ATOM NAMING AND ORDERING FOR HET GROUP
2IFBA 3
4 PLM TO FOLLOW PDB SPECIFICATIONS.
30 -APR-94.
2IFBA 4
1
131 ALA PHE ASP GLY T HR TRP LYS VAL ASP ARG ASN GLU ASN 2IFB 26
2
131 TYR GLU LYS PHE MET GLU LYS MET GLY ILE ASN VAL VAL 2IFB 27
2
3
1
2
1
2
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
continua…
40
O
GLY
4
2IFB
2IFB
2IFB
2IFB
2IFB
2IFB
38
39
40
41
42
43
IFABP
(intestinal fatty acid
binding protein)
(continuación del archivo PDB)
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
90.00 P 21
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
6.162
2.340 1.00 63.97
7.329
3.147 1.00 54.91
8.279
3.211 1.00 45.04
9.480
3.380 1.00 44.89
6.942
4.593 1.00 52.86
7.755
3.072 1.00 30.47
8.620
3.319 1.00 22.66
9. 093
2.072 1.00 24.99
9.827
2.119 1.00 26.08
0
6 1136
1
28
2
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
En el recuadro se muestran los valores de coordenadas atómicas (en Å)
La situación actual: un poco de estadística …
http://www.rcsb.org/pdb/statistics 28 febrero 2012
Yearly Growth of Total Structures in the PDB
El número total de estructuras sigue creciendo a ritmo (casi)
exponencial, pero …
http://www.rcsb.org/pdb/statistics 28 febrero 2012
el número de ‘folds’(modos de plegado) habría alcanzado un límite?
(el panorama según SCOP)
http://www.rcsb.org/pdb/statistics 28 febrero 2012
el número de ‘folds’ (modos de plegado) habría alcanzado un límite?
(el panorama según CATH)
http://www.rcsb.org/pdb/statistics 28 febrero 2012
Rayos X: no hay límite de MW:
piensen que se han cristalizado
virus enteros o el ribosoma!
NMR: limitado por el MW?
las nuevas tendencias apuntan
más a definir la dinámica que
la propia estructura
Estructuras por rayos X o por NMR?
http://www.rcsb.org/pdb/statistics 28 febrero 2012
Cristalografía de rayos X
Los pioneros: la cristalografía de rayos X
En 2009 se cumplieron 50 años de la primera
estructura de una proteína: la mioglobina
insulina
http://nobelprize.org/
Cristalografía de rayos X
Cristalografía de rayos X: una estación de trabajo
Cristalografía de rayos X:
la radiación sincrotrón
http://nobelprize.org/
http://nobelprize.org/
Resonancia magnética
nuclear (NMR)
Los pioneros: la resonancia magnética nuclear (NMR)
http://nobelprize.org/
Resonancia magnética nuclear (NMR):
la estructura y la dinámica de las proteínas
Resonancia magnética nuclear (NMR):
la estructura y la dinámica de las proteínas
Resonancia magnética nuclear (NMR):
la estructura y la dinámica de las proteínas
… o cómo construir un modelo
estructural a partir de:
- distancias entre átomos (NOEs),
-la orientación relativa de vectores
(las uniones químicas, RDCs)
y
- datos de desplazamiento químico (CSs)
La relajación de spins (ps-ns);
los acoples dipolares y escalares, los
desplazamientos químicos (ps-ms);
la dispersión de la relajación (s-ms);
la forma de las líneas, el intercambio
químico (ms-s);
y la técnica de “real time” NMR (s)
dan información sobre la dinámica de las
proteínas en escalas de tiempo muy
diversas
Movilidad relativa del esqueleto:
un núcleo ordenado y
extremos desordenados
Kurt Wüthrich
La proteína del prión:
coexistencia de
un dominio ordenado y
otro ‘nativamente’ desordenado
Kurt Wüthrich
El movimiento relativo del esqueleto
y de las cadenas laterales
Una visión del agua sobre
la superficie proteica
Kurt Wüthrich
Difracción de electrones,
microscopía electrónica
Los pioneros: la microscopía electrónica como herramienta
estructural y los complejos proteína-ácidos nucleicos
el nucleosoma
el método
el virus del
mosaico del
tabaco
http://nobelprize.org/
¿Cómo estudiar la reacción NU?
1.- Estudios en equilibrio
2.- Estudios cinéticos
(cinética rápida: típicamente en la escala
de miliseg)
Uso de agentes (físicos y químicos)
como perturbadores de la conformación:
Temperatura, presión hidrostática
Urea, cloruro/tiocianato de guanidinio
pH, fuerza iónica
Aditivos (p.ej. trifluoroetanol: TFE)
Técnicas experimentales
para estudiar la reacción NU
Espectroscopías
- Dicroismo circular
- Fluorescencia (intrínseca o sondas: p.ej. ANS)
- Absorción UV
- Resonancia magnética nuclear (NMR)
Determinación de forma y tamaño - Dispersión luminosa (scattering)
- Dispersión de rayos X (p.ej. SAXS)
- Exclusión molecular (p.ej. SEC-FPLC)
Alteración química
- Proteólisis limitada
+ SDS-PAGE, HPLC,
- Reactividad frente a agentes modificadores
ESI/MALDI-MS, NMR
- Intercambio H/D en uniones amida
Construcción de variantes - Mutagénesis dirigida a sitios
- Síntesis de péptidos
- Expresión de variantes truncadas, permutadas circularmente
- Complementación de fragmentos
Termodinámica
Estudios funcionales
- Microcalorimetría de titulación (ITC) y de barrido (DSC)
- Catálisis enzimática
- Unión de ligandos
¿Cómo estudiar la reacción NU?
1.- Estudios en equilibrio
2.- Estudios cinéticos
(cinética rápida: típicamente en la escala
de miliseg)
Uso de agentes (físicos y químicos)
como perturbadores de la conformación:
Temperatura, presión hidrostática
Urea, cloruro/tiocianato de guanidinio
pH, fuerza iónica
Aditivos (p.ej. trifluoroetanol: TFE)
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
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
The context where CD
becomes a useful tool
in biochemistry
The folding reaction
NU
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
An intuitive approach
to understand the nature
of polarized light and
its interaction with matter
Unpolarized, linearly (or plane)
polarized, and circularly polarized light
What is
optical
rotatory
dispersion
(ORD)?
=[]cl
nLnR
(Ó )
Two systems to represent a light beam
y
y
z
x
x
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 + wavelength (= +/2)
What would happen if the dephasing were instead - wavelength (= -/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?
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 + wavelength (= +/2)
What would happen if the dephasing were instead - wavelength (= -/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?
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 + wavelength (= +/2)
What would happen if the dephasing were instead - wavelength (= -/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?
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 + wavelength (= +/2)
What would happen if the dephasing were instead - wavelength (= -/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?
Electromagnetic waves and circular dichroism:
an animated tutorial
By András Szilágyi ([email protected])
www.enzim.hu/~szia/cddemo/edemo0.htm
Circular Dichroism (CD), a pictorial view
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 (,
theta), an angle parameter expressed in (mili)degree
units.
What is Circular
Dichroism (CD)?
Two equivalent
expressions:
ALAR
=[]cl
A=cl
Plane polarized
light turns into
elliptically
polarized light
by the
differential
absorption of
an optically
active
chromophore
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: R L)
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:
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
The ORD spectrum looks like the derivative (but it
is not) of the CD spectrum, however, the
dependence with 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
Physical conditions allowing the existence of optical activity
Optically
active
transition
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):
The (quasi)linear relationship existing between
molar ellipticity ([]) and the difference in the molar
extinction coefficients ()
Differential LambertBeer’s law
Definition of
absorbance A
Molar ellipticity ([
]) and the difference in the
molar extinction coefficients () are equivalent
measurements (convertible by a constant factor)
How come [
] = 3300 ?
The basic equations of ORD and CD
ORD
CD
The units:
ORD
O
cm2 dmol-1
CD
O
cm2 dmol-1
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!
The CD instrument: the spectropolarimeter
Pockels cell
Calibration:
CSA
-4.9 @ 192.5 nm
+2.36 @ 290.5 nm
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.
Practical aspects I:
Manufacturers: Horiba-Jobin Yvon, Jasco, AVIV
A ~ 10-4 A
More potent light sources vs. efficient
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 value), or by
quantitative amino acid analysis
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 ME < 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 < 190 nm)
How CD
becomes useful to
understand protein structure
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
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
The electronic transitions in proteins:
The peptide bond: n
* (br, w) ~ 210 nm
* (sh, s) ~ 190 nm
Cystine:
S
3
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)
… also prosthetic groups (e.g. heme) and
metalloproteins
Near UV region
(250-340 nm)
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
Circular Dichroism (CD)
50
ES-L
S126C S265C ES-L
S126C ES-L
S265C ES-L
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
Estimate secondary structure content
The reference
spectra (basis
set) for the
different types
of secondary
structure: helix, 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: helix,
parallel and antiparallel sheet, type I,
II and III turns
(Wetlaufer)
Spectral deconvolution into standard components
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 structure than on structure), and the
variable contribution of aromatic residues in this spectral region (see below)
All proteins
All
proteins
+ proteins
/ proteins
Disordered proteins
The contribution of aromatic residues
A cautionary note whenever interpreting the
contributions to []222!
Ligand binding: calcium binding to calmodulin
The binding of an intercalator molecule to dsDNA
Two coupled equilibria: the folding of protein
P and the binding of anionic ligands
Two structurally related proteins exhibiting
very different folding mechanisms:
bovine alpha-lactalbumin (
-LA) and lysozyme (HEWL)
HEWL
apo -LA
HEWL
apo -LA
CD reveals the
presence of
folding
intermediates:
-LA vs. HEWL
U
U
N
N
HEWL
MG -LA
(Kuwajima)
The ‘molten
globule’ (MG) state
of -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
-LA
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
The channel polypeptide
P190
changes its
conformation as a
function of pH
Folding kinetics detected
by CD
(time resolved CD)
The case of cytochrome c
(Elöve, Englander, Roder)
Folding kinetics
of
HEWL and -LA
(Kuwajima)
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
Reference books
1979
2005
1997
1996
2009
1984
1998
1980
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
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:
El instrumento de medida:
el espectropolarímetro
Celda de Pockels
La calibración del
espectropolarímetro
CSA
Rango A280 ~ 0.4-1.0 en proteína
-4.9 @ 192.5 nm
+2.36 @ 290.5 nm