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Lecture 5

Lecture 5 protein folding.docx

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Stavroula Andreopoulos

Lecture 5 Ainfinsen’s Experiment - proteins can fold reversibly - enzyme ribonuclease A from bovine pancreas was used to prove that all information needed to fold a polypeptide chain into its native structure is contained in its amino acid sequence - ribonuclease A – protein with 124 residues and four disulfide bonds - ribonuclease cleaves chains of ribonucleic acid - ribonuclease is an active enzyme only in its native form – loss of activity in a denaturation experiment was proof of loss of structure - experiment – treatment of ribonuclease solutions with a combination of urea and mercaptoethanol - urea – unfolds protein - mercaptoethanol – reduces disulfide bridges - treatment destroyed all enzymatic activity in ribonuclease A - removing mercaptoethanol but not urea restores about 1% of enzymatic activity – attributed to the formation of random disulfide bonds in the denatured protein - eight Cys residues in ribonuclease – 105 possible ways to create four disulfide bridges - simultaneous removal of both mercaptoethanol and urea result in full restoration of enzymatic activity – renaturation and correct formation of disulfide bridges - information needed for folding resides entirely within the amino acid sequence Mechanism for Protein Folding - mechanism stable formation of a protein is complex - Levinthal’s paradox – many conformations are possible for a typical protein that there is not sufficient time for the sampling of all conformations - hypothesize that proteins must fold by specific folding pathways - secondary structures (helices, sheets, turns) form first - nonpolar residues aggregate – hydrophobic collapse - formation of long range interactions between secondary structures or other hydrophobic interactions are subsequent - process of folding may involve one or more intermediate states – transition states known as molten globules - many proteins appear to possess small amounts of residual structure due to hydrophobic interactions even while denatured – strong interresidue contacts between side chains distant in native structure - sites of nucleation – interactions even while denatured, and small amounts of secondary structure - molten globule – flexible but compact form characterized by secondary structure, no precise tertiary structure, and a loosely packed hydrophobic core - for any given protein there may be multiple folding pathways - energy landscape – folding process as a funnel of free energies; at the top of the funnel represents many possible unfolded states of a polypeptide chain characterized by a high free energy and conformational entropy - polypeptides fall down the wall of the funnel as contacts between residues establish different folding possibilities; narrowing funnel represents smaller number of available states as the protein approaches its final state - bumps/pockets in funnel walls represent partially stable intermediates of the folding pathway - bottom of the funnel – most stable/native folded state of a protein Thermodynamic Driving Force for Folding Proteins - free energy change for the folding of a globular protein must be negative if the protein is approaching its most stable conformation - free energy change for folding depends of changes in enthalpy and entropy Tertiary Structure - typical folded protein is marginally stable - marginal stability is important for flexibility and motion - chemical bonds undergo a variety of motions – vibrations, rotations - many noncovalent interaction within a protein can be interrupted, broken, rearranged rapidly Motion in Globular Proteins - proteins are dynamic structures – oscillate and fluctuate – essential for protein function (ligand binding, enzyme catalysis, enzyme regulation) - atomic fluctuations – typically random and very fast, often occurring over small distances (vibra
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