BCHM461 EXAM 3 STUDY GUIDE Professor LaRonde Spring 2014 Chapter 4 The Three Dimensional Structure of Proteins I 4 4 Protein Denaturation and Folding Proteostasis the continual maintenance of the active set of cellular proteins required under a given set of conditions it requires o Coordinated function of pathways for protein synthesis and folding the refolding of proteins that are partially unfolded and the sequestration and degradation of proteins that have been irreversibly unfolded o Folding can occur spontaneously or by the help of chaperones misfolding of proteins can produce aggregation which leads to diabetes Parkinson or Alzheimer diseases Loss of protein structure results in loss of protein function o Denaturation a loss of three dimensional structure sufficient to cause loss of function Temperature high temperature causes protein destabilization pH disrupt hydrophobic interactions of the core of globular proteins disrupting hydrogen bonding Denaturing chaotropic reagents such as urea guanidine detergents etc Amino acid sequence determines tertiary structure o All molecules of a species have exactly specified sequences of amino acids o The tertiary structure of a globular protein is determined by its amino acid sequence Proof experiments showing that denaturation of some proteins is reversible certain globular proteins denatured by heat extreme pH or denaturing reagents will regain their native structure and their biological activity if returned to conditions in which the native conformation is stable renaturation Polypeptides fold rapidly by a stepwise process o Levinthal s paradox 100 amino acids protein where each amino acid can adopt 2 conformations This gives 2100 1 27 1030 possibilities If it takes 10 13 seconds to investigate each possibility it will take 4 109 years to test all possible structures o Three models proposed to provide pathways to folding The framework model proposed local elements of native secondary structure would form independently of tertiary structure These would diffuse and collide to form the correct tertiary structure The nucleation model proposed that some neighboring residues in sequence would form secondary structure and this would act as a nucleus for native structure formation The hydrophobic collapse model postulated that a protein would collapse rapidly around its hydrophobic side chains and rearrange from a conformation space restricted intermediate o The major folding pathways are hierarchical o Local secondary structures form first alpha helix or beta sheets Ionic interactions involving charged groups that are often near one another in the polypeptide chain help guide early folding steps Random formation of short stretches of secondary structure o Long range interactions between two elements of secondary structure form stable folded structures those regions adopting the native like secondary structure adhere to form clusters of secondary structure o Formation of the molten globule where regions of secondary structure are clustered in a close state to the native state but hydrophobic regions may still be exposed to solvent o Molten globule rearranges to give compact tertiary structure hydrophobic interactions aggregation of nonpolar amino acid side chains provide an entropic stabilization to intermediates and eventually to the final folded structure o Complete domains are formed larger proteins with multiple domains are synthesized and domains near the amino terminus which are synthesized first may fold before the entire polypeptide has been assembled o Unfolded states are characterized by a high degree of conformational entropy and high free energy Some proteins undergo assisted folding o Not all proteins fold spontaneously some need chaperones proteins that interact with partially folded or improperly folded polypeptides facilitating correct folding pathways or providing microenvironments in which folding can occur o Two major chaperone families Hsp70 and chaperonins Hsp70 protect both proteins subject to denaturation by heat and new peptide molecules being synthesized they also block the folding of certain proteins that must remain unfolded until they have been translocated across a membrane Chaperonins bind unfolded partly folded and incorrectly folded proteins but not proteins in their native state originally called heat shock proteins Hsp since their expression was induced by exposing cells to high temperature GroEL and GroES from E coli function together as a complex of 14 polypeptide chains of GroEL and 7 polypeptide chains of GroES o The folding pathways of some proteins require two enzymes that catalyze isomerization reactions Protein disulfide isomerase PDI catalyzes the interchange or shuffling of disulfide bonds until the bonds of the native conformation are formed catalyzes the elimination of folding intermediates with inappropriate disulfide cross links reduced PDI catalyzes the rearrangement of the non native disulfide bonds Peptide prolyl cis trans isomerase PPI catalyzes the interconversion of the cis and trans isomers of Pro residue peptide bonds Defects in protein folding provide the molecular basis for a wide range of human genetic disorders o Type 2 diabetes Alzheimer disease Huntington disease and Parkinson disease are associated with a misfolding mechanism a soluble protein that is normally secreted from the cell is secreted in a misfolded state and converted into an insoluble extracellular amyloid fiber disease amyloidosis Importance of structural studies o Basic science Mechanism enzymes ion channels molecular motors etc Role of mutations deletions in causing disease Complex formation protein protein protein DNA protein peptide etc Insights into role of proteins structural genomics o Medical commercial applications Rational drug design HIV protease kinase inhibitors Improve protein stability activity for commercial process o In order to full model the functioning of the cell Techniques used to solve the structures of proteins o X ray crystallography Advantages atomic resolution data nature of protein crystals allows the diffusion of small molecules into the crystal can study complexes in the Megadalton range Disadvantages requires large amounts of highly pure protein limitation on flexibility of molecule loops often invisible formation of crystals is unpredictable takes a lot of time Crystals comprised of millions of copies of the target macromolecule interactions between molecules are equivalent to