lec11-Denaturation-aggregation

lec11-Denaturation-aggregation - 7.88 Lecture Notes - 11...

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7.88 Lecture Notes - 11 7.24/7.88J/5.48J The Protein Folding Problem Protein Folding Intermediates and the Failure of Protein Folding Mutants affecting intermediates Protein Misfolding and Inclusion Body Formation A. Protein Folding Intermediates and the Failure of Protein Folding We discussed last week the refolding of cytochrome c, whose in vitro refolding pathway is robust. Considering proteins that successfully refold: Discovered that a key intermediate had a somewhat unexpected structure; N- and C- terminal helices docked, but interior fold not folded, or not-native-like. But if you are investigating the properties of cardiac muscle proteins, cytochrome c doesn’t help you; need to study the refolding of your protein!! What you might discover- is that you would clone gene for cardiac actin, purify cardiac actin Denature in urea, dialyze to refold, fail to obtain soluble actin: Well maybe purification regime does some subtle damage; Clone coding sequence into expression vector in E.coli; chain synthesized at high levels - but lo? fail to obtain soluble protein with properties of actin For many purified proteins, attempts to refold from the fully denatured state fails: No detectable renaturation - Of course generally these not reported; only successful cases reported. Very difficult, essentially impossible from scientific literature to determine if sequence sufficient to drive in vitro refolding o actin o tubulin o T4 DNA polymerase o Rubisco –ribulose bisphosphate decarboxylase o Collagen Yield low and highly variable (diluting from denaturant to buffer) o Beta galactosidase o Tryptophanase o Multi-subunit proteins: beta-galactosidase, lactic dehydrogenase
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B. How and Why do In Vitro Folding Reactions Fail?? This was examined systematically by a French group Michel Goldberg and Charles Ghelis and Jeanine Yon (authors of the best textbook on the subject) One of their early observations was that If remove denaturant by dialysis (slow) refolding fails: If remove denaturant by dilution (fast) higher refolding yield. Initial insight from this came from a two stage experiments - Suppose we do dilution out
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This note was uploaded on 02/21/2008 for the course BIOL 7.88 taught by Professor Gossard during the Fall '03 term at MIT.

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lec11-Denaturation-aggregation - 7.88 Lecture Notes - 11...

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