Lectures 5 & 6

Lectures 5 & 6 - Lectures 5 & 6 Chapter 3 &...

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Chapter 4 - Protein structures have evolved to function in particular cellular environments – conditions different from those in the cell can result in protein structural changes, large and small – a loss of 3-D structure sufficient to cause loss of function is called denaturation – most proteins can be denatured by heat, extremes of pH, by certain miscible organic solvents such as alcohol or acetone, by certain solutes such as urea and guanidine hydrochloride, or by detergents – each of these denaturing agents represents a relatively mild treatment in the sense that no covalent bonds in the polypeptide chain are broken – organic solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up the stable core of globular proteins - Tertiary structure of a globular protein is determined by its amino acid sequence. The most important proof of this came from experiments showing that denaturation of some proteins reversible. Certain globular proteins denatured by heat, extremes of pH, or denaturing reagents will regains their native structure and their biological activity if returned to conditions in which the native conformation is stable. This process is called renaturation. o Classic example – denaturation & renaturation of ribonuclease A, demonstrated by Christian Anfinsen in the 1950s – purified ribonuclease A denatures completely in a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the 4 disulfide bonds to yield 8 cys residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the entire polypeptide from its folded conformation. Denaturation of ribonuclease is accompanied by a complete loss of catalytic activity. When the urea and the reducing agent are removed, the randomly coiled denatured ribonuclease spontaneously refolds into its correct tertiary structure, w full restoration of its catalytic activity. (figure 4-26) urea denatures the ribonuclease, and mercaptoethanol reduces and thus cleaves the disulfide bonds to yield 8 Cys residues. Renaturation involves reestablishing the correct disulfide cross-links. The refolding of ribonuclease is so accurate that the 4 intrachain disulfide bonds are reformed in the same positions in the renatured molecule as in the native ribonuclease. o Calculated mathematically the 8 Cys residues could recombine at random to form up to 4 disulfide bonds in 105 diff ways. In fact, an essentially random distribution of disulfide bonds is obtained when the disulfides are allowed to re-form in the presence of denaturant (without reducing agent), indicating that weak bonding interactions are required for correct positioning of disulfide bonds and restoration of the native conformation. -
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Lectures 5 & 6 - Lectures 5 & 6 Chapter 3 &...

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