MCB110L_lecture16

MCB110L_lecture16 - MCB110L Lecture 16 03/11/11 Andreas...

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MCB110L Lecture 16 03/11/11 Andreas Martin
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Protein absorbance and fluorescence spectroscopy Absorbance A = -lg I/I 0 = ε c l Lambert-Beer Law c
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UV absorbance = excitation of electrons absorbance spectra: fine structure broadened different intensities Excited states Ground state reason: variety of possible transitions (vibrational and rotational states) 2-atomic molecule
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e - transitions upon photon absorbance: - electronic states S 0 –S 1 ~ 80 kcal/mol - vibrational states ~ 10 kcal/mol - rotational states ~ 1 kcal/mol E = N h ν = N h c/ λ Δ E = 80 kcal/mol -> λ = 360 nm Return to ground state: energy transformed into rotational and vibrational energy of surrounding molecules (solvent), = release of heat Fluorescence, Phosphorescence chemical reaction (photoreaction) Absorbance of biomolecules: “phenomenology”, cannot be described by discrete E transitions solvent = H 2 O, -> strong interactions, -> band broadening, λ < 170 nm not possible
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Source: Brown: An Introduction to Spectroscopy for Biochemists n -> π * λ = 210-220 nm ε = 100 M -1 cm -1 π -> π * λ = 190 nm ε = 7000 M -1 cm -1 proteins: absorbance by peptide bond and side chains
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Absorbance of a protein: Lysozyme aromatic amino acids and backbone „aromatic range“
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Absorbance of aromatic amino acids Trp Tyr Phe (10-fold enhanced)!) Trp Tyr Source: van Holde et al.: Physical Biochemistry => strong absorbance in far-UV! π π * transitions (1 mM) (0.1 mM) (0.1 mM)
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(1 mM) (0.1 mM) (0.1 mM) Absorbance of aromatic amino acids
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Dependence of absorbance on the solvent / environment: differential solvation or stabilization of ground and excited states (e.g. π and π *) causes: - λ -shift - Δ ε - broadening of absorbance bands transfer of a chromophore polar <-> apolar environment (e.g. during protein unfolding) leads blue or red-shift of absorbance bands -> difference spectra with distinct maxima absorbance can be used as a probe to follow protein folding / unfolding reactions example: red deer RNase (no Trp, only Tyr) difference spectrum N-U blue-shift biggest Δ A in the flank of the spectrum (287 nm)
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RNase T1: absorbance and changes upon unfolding N N U U N - U N - U Wild type: 1 Trp 9 Tyr W59Y- mutant: 10 Tyr 15 µ M RNase T1 0.1 M NaAc, pH 5.0 25°C, +/- 6.0 M GdmCl Tyr, 287 nm Trp, 291 nm Source: Schmid: Optical spectroscopy to characterize protein conformation In: Creighton: Protein Structure, a practical approach, IRL Press, 1997 Molar absorbance (M -1 cm ) important: Trp: no absorbance >310 nm Tyr: no absorbance >300 nm
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RNase T1: Thermal and GdmCl-induced unfolding [GdmCl] (M) Absorbance as a probe to monitor conformational changes and unfolding transitions of proteins native unfolded native unfolded important: denaturants (e.g. GdmCl) can affect absorbance (solvent polarity , red -shift of bands)
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absorbance spectrum Fluorescence emission : Why only one band?? Why shifted to longer wavelengths?
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This note was uploaded on 09/12/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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MCB110L_lecture16 - MCB110L Lecture 16 03/11/11 Andreas...

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