Lab 5: Fluorescence Quenching-Questions - Optical Spectroscopy | PHYS 552, Lab Reports of Optics

Download Lab 5: Fluorescence Quenching-Questions - Optical Spectroscopy | PHYS 552 and more Lab Reports Optics in PDF only on Docsity! Physics 552 Optical Spectroscopy (Fall 08) - 7 - Lab 5: Fluorescence Quenching - Questions Experiment I - Quenching of Tryptophan Fluorescence in Lysozyme by Acrylamide – Fractional Accessibility to Quenchers For the first part of the lab, we wish to explore one way to use fluorescence quenching to study protein structure. Lysozyme is a protein, found e.g., in secretions such as tears, as well as in egg white. It contains 6 residues of the fluorescent amino acid tryptophan; 4 are exposed and 2 are buried inside the protein. • For a good reference, see Eftink and Ghiron, “Exposure of Tryptophanyl Residues in Proteins. Quantitative Determination by Fluorescence Quenching Studies”, Biochemistry 15 (1976) 672. • You can check out the lysozyme structure at http://www.rcsb.org/pdb/explore.do?structureId=2HS9 Here you can use the various display options such as WebMol or JMol to highlight the tryptophan residues and see which are buried and which are exposed. Here is a three dimension plot of the protein structure of chicken egg white lysozyme. The whole protein has been drawn using a van der Waals surface. Each tryptophan residue has then been drawn with the bonds drawing method using an exaggerated radius. The yellow and blue tryptophan residues are buried while the other 4 are exposed. Physics 552 Optical Spectroscopy (Fall 08) - 8 - The solutions we used for our experiments were chicken egg lysozyme (from Sigma Aldrich) in 10mM TRIS buffer (pH 8.0). We then titrated in acrylamide to observe the dynamic quenching. (Q1) a) Turn in a plot of the absorption spectra versus wavelength. b) Identify on the plot the contribution to the absorption spectra from the tryptophan and from the acrylamide. 3d structure of chicken egg-white lysozyme showing the tryptophan residues. This structure has been rendered using VMD [Humphrey, W., Dalke, A. and Schulten, K., "VMD - Visual Molecular Dynamics", J. Molec. Graphics, 1996, vol. 14, pp. 33-38. http://www.ks.uiuc.edu/Research/vmd/] The PDB file used is 2HS9 [Von Dreele, R.B. Multipattern Rietveld refinement with protein powder data: an approach to higher resolution To be Published, http://www.rcsb.org/pdb/explore.do?structureId=2HS9] Physics 552 Optical Spectroscopy (Fall 08) - 11 - Experiment II - Ionic Quenching of Fluorescein by Iodide The data consists of the 10 intensity values that you recorded for the 4 sets of 6 samples. You will prepare a Stern-Volmer plot for each of the 4 sets of data. That is, a plot I0/I vs. [Q]. For each Stern-Volmer plot, perform a linear fit with the intercept set to 1.0. Your data files will have three columns: Iteration Intensity IntensityStdError 1 43239.6 316.26 2 43584.9718 324.29 3 43374.0928 357.32 4 43346.2571 309.79 … … … Use the average of the 10 intensity values for your plots and fits. The intensity values already include an excitation side instrumentation correction (this was set when you checked the “ratio with excitation” box in the software). Q5) Turn in a graph showing the KI and ICH data sets. The KI data set should not be linear. Why does the data show an upward curvature? The ICH samples should be similar to the KI data set, but be more linear. Why should this be true? Q6) The last two data sets (ICA and ICB) correspond to low concentrations of KI. Turn in a graph of these two data sets and include the values of Ksv in the plot. Why does Ksv increase with increased ionic strength? Because of the small steps and overall low iodide concentration for these set of samples, we wind up expecting less than 3% changes in intensity per step by samples 4 and 5. Try to get the best fits you can for your Stern-Volmer constants. Physics 552 Optical Spectroscopy (Fall 08) - 12 - *For 0 m α = , equation 6 of the inner filter appendix becomes: ( ) ( )3 0 , , expd F F xd N I p x y z q x dx dy dzα α= − [6*] 7. With the same assumptions as in the appendix, this becomes: 0 2 exp sinh 2 2 y z F x x x x d Ins F x LN I q α α αβ α Δ Δ Δ⎛ ⎞ ⎛ ⎞= −⎜ ⎟ ⎜ ⎟ ⎝ ⎠ ⎝ ⎠ [7*] 8. Using the same limits, the number of photons Nd0 detected in this limit is given by the same expression as before: zyxFFInsd qIN ΔΔΔ= αβ0 0 [8] 9. Taking the ratio of equations [8] and [7*], we can derive correction factors to apply to the measured fluorescence spectra that remove the non-linear effects in the signal on the excitation side. 0 2exp 2 sinh 2 x x x x d d x x LN N α α α ⎛ ⎞Δ ⎜ ⎟⎛ ⎞⎜ ⎟= ⎜ ⎟ Δ⎛ ⎞⎜ ⎟⎝ ⎠ ⎜ ⎟⎜ ⎟⎝ ⎠⎝ ⎠ [9*] 10. Substituting ln(10) ( )exx x A L λα = : ( ) 2 ln(10) ( ) 210 ln(10) ( )sinh 2 ex ex x A x corr ex x x A LEm Em A L λ λ λ ⎛ ⎞ ⎜ ⎟ ⎝ ⎠ ⎛ ⎞Δ ⎜ ⎟ ⎜ ⎟= ⎜ ⎟⎛ ⎞Δ ⎜ ⎟⎜ ⎟⎜ ⎟⎝ ⎠⎝ ⎠