Luz linear Luz circular polarizada - USP - IFSC...– Equilibrium intermediates – Kinetics –...
Transcript of Luz linear Luz circular polarizada - USP - IFSC...– Equilibrium intermediates – Kinetics –...
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Luz polarizada
Luz linear
polarizada
Luz circular
polarizada
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φ λ= −180l n nL R( ) / graus
θ π= −2 303 180 4, ( ) /A AL R graus
Atividade óptica
• Mudança na velocidade de propagação das duas componentes: dispersãorotatória óptica (ORD) e birrefringência• Diferença na absorção das mesmas: dicroísmo circular e elipticidade.
A rotação óptica como função do comprimento de onda é chamada de dispersão rotatória óptica (ORD)
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Dicroísmo circular e birrefringência
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Interações: • pelas mudanças nas velocidades de propagação dos raios através da amostra que tem relação com o índice de refração do meio = Dispersão Óptica Rotatória (ORD);
•pela diferença da intensidade de absorção dos raios através da amostra = Dicroísmo Circular (CD).
Lembrando: A = ε C lCD seria ∆A = Al - Ar = εl C I - εr C I =∆ ε C I ⇒⇒⇒⇒ CD= ∆ε =εl - εr
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clA ε=
( ) Acl ∆=∆ /1ε
Onde ∆ε é definido como εl - εr
( ) ( ) ( ) ( )rlrl EEEEtgrad +−=≈ θθ
−
+
−
−
−
−
= expexpexpexp // rr AAAA
Desta forma, temos:
−
+
−
−
−
−
= 2exp2exp2exp2exp // rr AAAA
Expandindo as exponenciais e convertendo em graus, temos:
( ) πθ 4.10ln.180 Agraus ∆=
A∆= 98,32θ
Esta elipticidade é proporcional ao CD de forma que :
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Por razões históricas, a concentração era medida em g/100 cm3, a elipticidade molarera medida em grau.cm2.g-1.
Para corrigir estas unidades temos que multiplicar a equação anterior por 100.
e
[ ] clθθ .100=
[ ] ( )clA∆= .98,32.100θ
[ ] εθ ∆= .3298 units: deg cm2 dmole -1
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Cristal pizoelétrico
Esquema básico de um espectropolarímetro
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ASSIMETRIAS QUE PODEM LEVAR À ATIVIDADE ÓPTICANA ESTRUTURA DE PROTEÍNAS
•os aminoácidos (com exceção da Glicina) são compostos assimétricos
•estrutura primária é inerentemente assimétrica devido as ligações peptídicas que são opticamente ativas:
•transições eletrônicas no grupo amida:•transições eletrônicas no grupo amida:
•n-π* (210 - 230 nm)•π-π* (180 - 200 nm)
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Secondary Structure & CDα-helix β-sheet turn
12
3 4
ββββ-sheet
αααα-helix
other/PII
turns
PolyProII (PII)
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CD Spectrum →→→→ Structure Linear relation Cλ = Σ fk Bkλ
Cλ: protein CD spectrum; Bkλ: component secondary structure spectra; fk is the fraction of the secondary structure k.
Determination of Bkλ model polypeptides or set of proteins.
Curve Assumptions
1. CD contributions from individual secondary structures are additive
2. The ensemble-averaged solution structure and the time-averaged solid-state structure are equivalent
3. CD contributions from non-peptide chromophores do not influence the analysis
4. The effect of the tertiary structure on CD is negligible
5. Effects of the geometric variability of the secondary structures are not explicitly considered
Curve Fitting
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Secondary Structure - Structural Decomposition
α-helixA
β-sheetB
TurnsT
C147
N1
X-ray Structure
fα 0.32
fβ 0.22
fT 0.26
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Methods for Protein CD Analysis
AlgorithmsVariable Selection
Locally Linearized Model
Self-consistent Method
Minimal Basis
CDProSoftware Package
MethodsLeast Squares Minimization
Ridge Regression
Singular Value Decomposition
Principal Component/Factor Analysis
Neural Networks
Convex Constraint Analysis
ProgramsSelcon3 CDsstr Contin
varselc, CDNN, K2D, CCA
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Protein Structure & CD Spectrum
αβαβαβαβ
αααααααα ββββββββα/βα/βα/βα/β α+βα+βα+βα+β
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Summary
Characteristic CD Spectra of Protein Secondary Structures
Different spectral regions give different structural information
The information content depends on the chromophore
Structure ���� Spectrum Spectrum ���� Structure
Chromophores Database of Structure/Spectra
Theoretical Methods Mathematical AnalysisTheoretical Methods Mathematical Analysis
Protein CD analysis
Secondary Structure Fractions
Number of α and β segments
Tertiary Structure Class
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APPLICATIONS OF CD TO PROTEINS
• Protein structure and function– Secondary structure analysis– Monitoring conformational change– Ligand binding
• Protein folding• Protein folding– Equilibrium intermediates– Kinetics– Stability of short helices, β-strands
• Comparison of mutant and wild-type– Secondary structure – Stability