lec10 - MIT OpenCourseWare http://ocw.mit.edu 7.88J Protein...

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Unformatted text preview: 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 . 7.88 Lecture Notes - 10 7.24/7.88J/5.48J The Protein Folding Problem Among non S-S proteins, one of those studied most intensively has been cytochrome c. Cytochrome c structure Equilibrium unfolding/refolding and kinetic refolding studies Hydrogen/Deuterium Exchange Roder and Eloves experiments to characterize the early intermediate A. Cytochrome c - Central protein in electron transport pathway Highly conserved through animal and plant kingdoms Within mitochondrial membrane Transparency of 3 species: Courtesy of Annual Reviews. Figure 6.31: The similar folded conformations of distantly related cytochromes c.: Salemme FR. "Structure and function of cytochromes c." Annu Rev Biochem. 1977;46:299-329. Figure 10.0: The Similar Folded Conformations of Distantly Related Cytochromes c (from Salemme, F. R. "Structure and function of cytochromes c." Annu Rev Biochem 46 (1977): 299-329.) Receives electron from cytochrome a/cytochrome c1 complex = cytochrome reductase; Passes it to cytochrome a/a3 = cytochrome oxidase; Alternates between oxidized (Fe+3) and reduced (Fe+2) states; Structure: Reduced state: 104 amino acids, basic (17 lysines) No beta sheet; two well formed alpha helices: 1>11; 89>101 Rest of chain is kind of wrapped around heme group Heme covalently linked to cysteine 14 and cysteine 17. In native state heme is liganded by Histidine 18 and Methionine 80. In hemoglobin the Fe heme complex binds oxygen; in cytochrome it binds an electron, because the 6th coordinating site of the Iron that would bind O2 is occupied by a methionine side chain. Yeast has two cytochrome cs. called iso 1 and iso 2 , which differ at several places in sequence. Iso-2 lacks free cysteine thiols, so not plagued by adventitious S-S bonds. Exploration of Protein Database Structure: B. Refolding of cytochrome c Nall, Barry, and Terry Landers. Guanidine HCl Induced Unfolding of Yeast Iso-2 Cytochrome c. Biochemistry 20 (1981): 5403-5411. Equilibrium denaturation as function of GuHCl concentration; Follow two uv absorption: A287 (tryp/tyr) and A 418 = heme group. Coincident with D 1/2 around 1 Molar Axes: Extinction coefficient vs. GdnHCL (pH 7.2, 20 o C) Looks two state, and seems to be concerted, aromatics exposed in same curve as heme. They calculate Gibbs Free energy of 3.1kcal/mole, 20C, extrapolated to 0 denaturant concentration. Now lets carry out kinetic experiment; Optical signal on left; 2 seconds/division on horizontal axis Clearly two kinetic phases; fast phase, all done by 5 seconds, then slower phase time constant about 100 seconds Similar kinetic phases regardless of which signal monitor, so not simply difference in heme relationship and overall conformation; So indicates the presence of some kinetic intermediate in the folding pathway:...
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lec10 - MIT OpenCourseWare http://ocw.mit.edu 7.88J Protein...

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