Principles of circular dichroism (CD) and its applications to proteins

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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

April 2016

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¿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

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La cadena polipeptídica como sucesión de planos

conectados por los vértices, relacionados a través de

las torsiones phi y psi

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El diagrama de Ramachandran

En la cadena polipeptídica plegada, los pares phi/psi adoptan sólo valores característicos que definen los diferentes tipos de estructura secundaria: alpha, beta y otras

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Las cadenas laterales de los aminoácidos también adoptan conformaciones características → los ángulos de torsión chi (chi1, chi2, etc.)

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Los ángulos de torsión chi adoptan valores característicos de acuerdo con el tipo de aminoácido (p.ej. Leu)

→ existen bibliotecas de rotámeros

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¿Cómo estudiar la reacción N↔U?

1.- Estudios en equilibrio

2.- Estudios cinéticos

(cinética rápida: típicamente en la escala de miliseg)

↓

Uso de agentes perturbadores de la conformación (físicos o químicos) :

Temperatura, presión hidrostática Urea, cloruro/tiocianato de guanidinio

pH, fuerza iónica

Aditivos (p.ej. trifluoroetanol: TFE)

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Técnicas experimentales

para estudiar la reacción N↔U

Espectroscopías - Dicroísmo 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 - Microcalorimetría de titulación (ITC) y de barrido (DSC) Estudios funcionales - Catálisis enzimática

- Unión de ligandos

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Técnicas experimentales

para estudiar la reacción N↔U

Cada una de ellas provee una señal característica que contiene información sobre la naturaleza y la

abundancia relativa de las especies involucradas en la reacción

A través de la medida de esta señal en cada muestra donde el equilibrio N↔U se haya perturbado, se puede inferir la presencia y cuantificar las especies

Con los datos obtenidos es posible obtener el valor de

las constantes termodinámicas de la reacción (K

_eq

, ΔG,

ΔH, ΔS)

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The context where CD becomes a useful tool

in biochemistry

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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

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An intuitive approach to understand the nature

of polarized light and

its interaction with matter

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The nature of polarized light

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What is optical rotatory dispersion (ORD)?

α⁽^Ó Φ⁾ α=[α]cl

n_L≠n_R

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Two systems to represent a light beam

x y

z x

y

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Three simple exercises to intuitively understand the (general) nature of polarized light and the CD phenomenon

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)!

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?

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?

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Three simple exercises to intuitively understand the (general) nature of polarized light and the CD phenomenon

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Three simple exercises to intuitively understand the (general) nature of polarized light and the CD phenomenon

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The interaction of polarized

light with matter

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Three simple exercises to intuitively understand the (general) nature of polarized light and the CD phenomenon

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Electromagnetic waves and circular dichroism:

an animated tutorial

By András Szilágyi ([email protected])

www.enzim.hu/~szia/cddemo/edemo0.htm

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Circular Dichroism (CD), a pictorial view

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However, both ORD and CD are different outcomes of the same physical phenomenon, i.e. the interaction of polarized light (E_R and E_L) 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 n_R ≠ n_L)

In CD, the detection consists in evaluating the change in the amplitudes (|E_R| and |E_L|) of the beams (through the change in absorption:

ε

_R^≠

ε

_L⁾

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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:

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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)

Uses of CD

Estimate secondary structure content Detect conformational changes

Measure ligand binding

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The ORD spectrum looks like the derivative (but it is not) of the CD spectrum, however, the

dependence with λ is different

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

For this reason, it is possible to measure optical activity in regions far from the absorption maximum (e.g. in sugars)

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The (quasi)linear relationship existing between

molar ellipticity ([Θ]) and the difference in the molar extinction coefficients (Δε)

Differential Lambert- Beer’s law

Definition of absorbance A

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How come [Θ] = 3300 Δ

ε

^?

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The instrumentation for measuring CD:

the spectropolarimeter

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•!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!

The CD instrument: the spectropolarimeter

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!

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Pockels cell

Calibration:

$* +2.36 @ 290.5 nm

$* -4.9 @ 192.5 nm CSA

The CD instrument: the spectropolarimeter

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Practical aspects I:

Manufacturers: Horiba-Jobin Yvon, Jasco, AVIV, Applied Photophysics (Chirascan models)

ΔA ~ 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 ε value), or by quantitative amino acid analysis

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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)

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How CD

becomes useful to

understand protein structure

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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

Main uses

Estimate the secondary structure content of a protein Detect conformational changes

Measure ligand binding

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The electronic transitions in proteins:

The peptide bond: n→π* (br, w) ~ 210 nm

π→ π* (sh, s) ~ 190 nm Cystine:

S S

χ₃

…and the aromatic residues (see below)

Far UV region (180-250 nm)

Near UV region (250-340 nm)

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

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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) (CD)

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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

-150 -100 -50 0 50

250 260 270 280 290 300 310 320 330 ES-bL

S126CS265CES-bL S126CES-bL

S265CES-bL

-14 0 14 28 42

250 260 270 280 290 300 310 320 0M,WT 0.0M,trunc. 2.0M,trunc. 5.0M,trunc. 6.6M,trunc. 6.6M,WT

Wavelength (nm)

Circular Dichroism (CD)

Wavelength (nm)

Javier Santos

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CD spectra of real

peptides and proteins

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All

α

^proteins

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All

β

^proteins

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α + β

^proteins

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α/β

^proteins

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Disordered proteins

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The contribution of aromatic residues

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A cautionary note whenever interpreting the contributions to [θ]₂₂₂!

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Deconvolution of CD spectra

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The reference spectra (basis set) for the

different types of secondary

structure: α helix, β sheet, and random coil

Estimate secondary structure content

A critical point is the wise choice of

standards

Based on known 3D structures taken from the PDB: α helix, parallel and anti-

parallel β sheet, type I, II and III β turns

(Wetlaufer)

Based on amino acid polymers: poly-K, poly-E (Fasman)

Problem: dependency on the length of helices, sheets or coils, uncertain contribution of turns

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Spectral deconvolution into standard components

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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)

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A collection of practical examples

of CD in structural biology

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Two structurally related proteins exhibiting very different folding mechanisms:

bovine alpha-lactalbumin (α-LA) and lysozyme (HEWL)

apo α-LA HEWL

HEWL

Apo α-LA

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Lisozima α-Lactalbumina

Dos proteínas estructuralmente muy similares

presentan transiciones N/U muy diferentes

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Desplegado de una proteína en el equilibrio

derivación del cambio de energía libre de la reacción (‘two states’)

N

U

k_u

k_f =

[U] k_u

[N] = k_f K_NU

ΔG⁰_{NU =}- RTln K_NU

[GdmHCl] (M)

Δ G

⁰_NU

= Δ G

_NU^{0 H}²^O

+ m[D]

[GdmHCl] (M) 1 2 3 4 5 6 ΔG_N→⁰ _U

f_D+f_N=1

El plegado/desplegado de una proteína es un fenómeno altamente cooperativo →

este hecho se refleja en el valor de la pendiente de la curva (m)

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HEWL

HEWL α-LA

α-LA

CD reveals the presence of folding

intermediates:

α-LA vs. HEWL

(Kuwajima)

N U

U/MG

N

N U

MG

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

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CD as a useful signal to

monitor folding kinetics

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Folding kinetics detected by CD

(time resolved CD)

The case of cytochrome c (Elöve, Englander, Roder)

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Folding kinetics of

HEWL and α-LA

(Kuwajima)

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Binding of an ‘invisible’ ligand to a protein:

Ca ²⁺ binding to calmodulin

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CD displays exquisite

sensitivity to the nature of the

binding process

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The binding of an intercalator molecule to dsDNA

Three binding modes

A change in spectral shape points to two different binding modes

Intercalation Major groove binding

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Coupling binding to folding:

the protein associated with the

ribozyme ribonuclease P

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Two coupled equilibria: ‘natively unfolded’ protein P folds upon binding of anionic ligands

Unfolded

+ SO₄^2-

dCTP

CMP

formate dCMP

Buffer cacodylate does not bind to the protein Tyr environment

K_fold and K_a cannot be determined independently

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A physiological temperature- induced conformational

transition of IFABP enhancing

fatty acid binding

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IFABP

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IFABP

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IFABP

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Protein conformational plasticity:

fatty acid binding modulates the fine structure of an abridged form of

IFABP

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Consolidation of a native fold by peptide recognition:

the case of E. coli thioredoxin

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C N

Build-up of a native-like near-UV CD spectrum upon complexation

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Recovery of the thermally-induced conformational transition upon complexation

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Brief introduction, tutorial with examples and programs:

www.imb-jena.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:

www-structure.llnl.gov/cd/cdtutorial.htm

Some sites of interest on circular dichroism (CD):

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1996

1998 1984

1997 2005

1979

Reference books

1980 2009

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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

'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

‘Using circular dichroism spectra to estimate protein secondary structure’

Nat Protoc. 2006 ; 1(6): 2876-2890

‘Analysis of the kinetics of folding of proteins and peptides using circular dichroism’

Nat Protoc. 2006 ; 1(6): 2891-2899

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Extra slides

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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:

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El instrumento de medida:

el espectropolarímetro

Celda de Pockels

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La calibración del espectropolarímetro

$* +2.36 @ 290.5 nm

$* -4.9 @ 192.5 nm CSA

Rango A₂₈₀ ~ 0.4-1.0 en proteína

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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 7 pH 7

pH 2

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The channel polypeptide P190

changes its

conformation as a function of pH

Principles of circular dichroism (CD) and its applications to proteins