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Lecture 8

BCH2011: Textbook summary - Lecture 8

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Monash University

LECTURE 8 Protein Denaturation and Folding: A native protein conformation is only marginally stable. In addition, most proteins must maintain conformational flexibility to function. The continual maintenance of the active set of cellular proteins required under a given set of conditions is called proteostasis. Cellular proteostasis requires the coordinated function of pathways for protein synthesis and folding, the refolding of proteins that are partially folded, and the sequestration and degradation of proteins that have been irreversibly unfolded. The marginal stability of most proteins can produce a tenuous balance between folded and unfolded states. As proteins are synthesized on ribosomes, they must fold into their native conformations. Sometimes this occurs spontaneously, but more often it occurs with the assistance of specialized enzymes and complexes called chaperones. Many of these same folding helpers function to refold proteins that become transiently unfolded. Proteins that are not properly folded often have exposed hydrophobic surfaces that render them ‘sticky’, leading to the formation of inactive aggregates. These aggregates may lack their normal function but are not inert. All cells have elaborate pathways for recycling and/or degrading proteins that are irreversibly misfolded. Loss of Protein Structure Results in Loss of Function: Conditions different from those in the cell can result in protein structural changes, large and small. A loss of 3D structure sufficient to cause loss of function is called denaturation. The denatured state does not necessarily equate with complete unfolding of the protein and randomization of conformation. Under most conditions, denatured proteins exist in a set of partially folded states. Most proteins can be denatured by heat, which has complex effects on many weak interactions in a protein (primarily on H-bonds). If the temperature is increased slowly, a protein’s conformation generally remains intact until an abrupt loss of structure (and function) occurs over a narrow temperature range. The abruptness of the change suggests that unfolding is a cooperative process: loss of structure in one part of the protein destabilizes other parts. Proteins can also be denatured by extremes of pH, by certain miscible organic solvents such as alcohol or acetone. 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; urea also disrupts hydrogen bond; extremes of pH alter the net charge on the protein, causing electrostatic repulsion and the disruption of some hydrogen bonding. The denatured structures resulting from these various treatments are not necessarily the same. Denaturation often leads to protein precipitation, a consequence of protein aggregate formation as exposed hydrophobic surfaces associate. The aggregates are often highly disordered. Amino Acid Sequence Determines Tertiary Structure: Denaturation of some proteins is reversible. Certain globular proteins denatured by heat, extremes of pH, or denaturing reagents will regain their native structure and their biological activity if returned to conditions in which the native conformation is stable. This process is called renaturation. A classic example is the denaturation and renaturation of ribonuclease. Purified ribonuclease A denatures completely in a concentrated urea solution in the presence of a reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight 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, with full restorating of its catalytic activity. The refolding of ribonuclease is so accurate that the four intrachain disulfide bonds are re-formed in the same positions in the renatured molecule as in the native ribonuclease. The Anfinsen experiment provided the first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, 3D structure. Subsequent work has shown that only a minority of proteins, many of them small and inherently stable, will fold spontaneously into their native form. Even though all protein
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