lec09 - MIT OpenCourseWare http/ocw.mit.edu 7.88J Protein...

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MIT OpenCourseWare http://ocw.mit.edu 7.88J Protein Folding Problem Fall 2007 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7.88 Lecture Notes - 9 7.24/7.88J/5.48J The Protein Folding Problem Fluorescence spectroscopy Denaturation and Denaturing agents Denatured State as a random coil (First Approx.) Renaturation/Refolding Protocols Detection of partially folded Intermediates So far we have examined the experimental evidence for three cases Ribonuclease S Heptad Repeat protein -Tropomyosin ; Coil <> helix transition in aqueous solution: S-peptide coil >helix transition in solution However, as we noted earlier the 22,000 + proteins in the protein database are primarily soluble globular proteins; And probably more than half to 3/4 of the proteins encoded by genomes are in this class: So need to understand how sequence drives fold, or to be A probably the majority of proteins encoded Methods for the study of the conformation of folding intermediates A. Fluorescence Spectroscopy J. R. Lakowicz Ch 23 pp518-567 Many molecules that absorb light in the UV or visible spectra, also re-emit photons, at longer wavelength: Fluorescence Phe Tyr and Tryp absorb in the region of: Amino Acid Abs max (nm) Molar absorbance M -1 cm -1 Phe 257.4 197 Tyr 274.6 1420 Tryp 279.8 5600
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10 (I o /I): I o is transmittance by reference solution, I is transmittance by sample. Molar absorbance is = A/bc, where b is path length in centimeters, and c is concentration in moles/liter 280mu. Phe peak is about 255 but 1/20 that of tryp peak at 280. Absorption in this region of the spectrum is due to changes in electronic energy levels, levels of orbital electrons. Photon absorption occurs in 10(-15) seconds, so short that nucleus doesn't change much in time interval; so goes up in energy level almost instantaneously , but lifetime is much longer, 10(-8) seconds. Return to ground state usually multi-step: small losses of energy in atomic collisions >> Goes back to lowest vibrational energy level state of excited electronic state (faster than emission); If molecule doesn't blow up, then after short period of time, returns to lowest energy level emitting a photon; since excited state has lost a little energy, then photon emitted will be lower energy, longer wavelength than photon absorbed; Lambda max for fluorescence: Phe 282 0.04 Tyr 303 0.21 Tryp 340 0.20 Emission occurs on nanosecond time scale; Process occurs like radioactive decay I = I o e (-t/g) , where t is time after end of light, g is average lifetime of fluorescent state; Because emission is a property of valence electrons, sensitive to environment; Fluorescence; tryptophane: in water 350; buried in proteins 330. Native florescent spectrum
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This note was uploaded on 11/11/2011 for the course BIO 7.344 taught by Professor Bobsauer during the Spring '08 term at MIT.

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lec09 - MIT OpenCourseWare http/ocw.mit.edu 7.88J Protein...

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