BCHM461 EXAM 3 STUDY GUIDE- Professor LaRonde Spring 2014Chapter 4- The Three-Dimensional Structure of ProteinsI. 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 unfoldedo 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 functiono 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 structureo All molecules of a species have exactly specified sequences of amino acidso 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 processo 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 structureso 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 hierarchicalo 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 structureo 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 structureo 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 solvento 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 structureo 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 foldingo 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 occuro 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 GroESo 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 conformationare 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 disorderso 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 studieso 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 processo In order to full model the functioning of the cell- Techniques used to solve the structures of proteinso 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