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Transcript
Rotation of Membrane Proteins : Determining Correlation Time •Fluorescence/Phosphorescence •Saturation Transfer EPR Hydrophobicity Scales of Amino Acids: Wimley and White Hydrophobicity Measurements 3 papers: Anal Biochem.213, 213-217 (1993) Biochemistry 35, 5109-5124 (1996) Nature Struc Bio 3, 842-848 (1996) Fluorescence anisotropy measurements are useful for labeled proteins in solution, but what about proteins imbedded into the bilayer? Recall: Rotational diffusion can only be measured if a significant amount of rotation occurs during the lifetime of the excited state. Membrane imbedded proteins have correlation times as slow as ms to ms. ~ns Possible solutions to measuring slower correlation times: 1) Utilize the triplet state (optical spectroscopy) 2) Saturation transfer EPR Rotational Diffusion constant for protein in membrane DR = kT/ (4pa2hh) Saturation Transfer EPR Hemoglobin in glycerol water is used as standard Stokes-Einstein equation tR = Vnw/(kT) Amino Acid Hydrophobicity Scales Octanol/water(buffer) partitioning Water/LUV partitioning Equilibrium dialysis and HPLC Ac-WL-X-LL Ac-WLm Volume Fraction Partition Coefficients: KV = Pb/Po KV = (Vo/Vb)Pb/(Ps-Pb) Pb = peptide in buffer Po = peptide in octanol Ps = stock peptide solution concentration Mole Fraction Partition Coefficients: Kx = Kv(vwat/voct) Where vwat/voct = 0.114 is the ratio of the molar volumes of water and octanol DG = -RT(lnKx)
