Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Rotation of Membrane Proteins : Determining Correlation Time•Fluorescence/Phosphorescence•Saturation Transfer EPRHydrophobicity Scales of Amino Acids:Wimley and White Hydrophobicity Measurements3 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 s to ms.~nsPossible solutions to measuring slower correlation times:1) Utilize the triplet state (optical spectroscopy)2) Saturation transfer EPRDR = kT/ (4a2h)Rotational Diffusion constant for protein in membraneSaturation Transfer EPRHemoglobin in glycerol water is used as standardStokes-Einstein equationR = Vnw/(kT)Amino Acid Hydrophobicity ScalesOctanol/water(buffer) partitioningWater/LUV partitioningEquilibrium dialysis and HPLCAc-WL-X-LLAc-WLmVolume Fraction Partition Coefficients:KV = Pb/PoKV = (Vo/Vb)Pb/(Ps-Pb)Pb = peptide in bufferPo = peptide in octanolPs = stock peptide solution concentrationMole Fraction Partition Coefficients:Kx = Kv(vwat/voct)Where vwat/voct = 0.114 is the ratio of the molar volumes of water and octanolG =
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