The Anfinsen experiment: spontaneous folding The seminal experiment reported by Anfinsen demonstrated that RNase A could be fully denatured by: 1. reducing the disulfide bonds (using B-mercaptoethanol) 2. dissolving in 8 M urea and then fully renatured on diluting out the urea and allowing the disulfides to reoxidize. Essentially full return of catalytic activity was obtained. This experiment demonstrated that all the information necessary to determine the three-dimensional fold was incorporated in the amino acid sequence. ‘Anfinsen (1973) Science, 181223 Anfinson's work on ribonuclease A eM UREA + REDUCING AGENT = RANDOM COIL NATIVE RIBONUCLEASE A [AND REDUCING SLOW RECOVERY OF ACTIVITY REFOLDINGWhy study protein folding? There are many diseases known to result from misfolding - Alzheimer's disease. - Cystic fibrosis. - Mad Cow disease. - Many cancers. Folding of peptide drugs is an important issue for biotechnology The prediction of secondary and tertiary structure from the primary sequence is important for interpretation of the genome. Protein misfolding Cells use chaperones to guide folding When misfolding occurs cells will hydrolyze the misfolded proteins. For biotechnology there is an important issue of understanding how folding occurs so that we can express proteins in a properly folded state.Aggregation Kinetic stability is a measure of how rapidly a protein unfolds. It isa particularly important consideration for proteins that unfold very slowly or denature irreversibly. This is indicated in the scheme below: Folded = Unfolded => Irreversibly Ky k, Denatured We are concerned here with irreversible aggregation associated with changes in protein structure. P22 tailspike protein ‘Tho carly 1980s saw one of the first serious investigations of protein misfolding. These studies focused on temperature- sensitive mutations (mutations allowing growth at 24 °C but not at 38°C ) in the tailspike protein of bacteriophage P22. The majority of the temperature-sensitive mutations they found, despite having only one amino avid altered, caused the tailspike protein to end up as insoluble gunk at high temperatures Since these folding failures were occurring in bacterial cells, that were growing in the laboratory, it was now possible to analyze what went wrong in a protein's folding process.Protein folding diseases There are a number of important diseases that occur due to misfolding of proteins. Examples include famial amyloidic polyneuropathy, Alzheimer’s disease, ete. There are two possible problems that can result from protein misfolding. First, the protein may not be produced in suflicient quantity because the misfolded protein has exposed amino acids that are easily hydrolyzed. ‘This type of problem occurs for the P22 tailspike mutations in nature. A second type of problem that can occur is aggregation. The misfolded protein can form a deposit or plaque (or gunk as it is known in the science community). We consider next the consequence of mutatation in transthyretin. Transthyretin is a protein that transports the thyroid hormone. At least 50 single point mutations in transthyretin result in misfolding that causes aggregation, As the protein is being broken down, it forms insoluble gunk, and the insoluble gunk poisons the tissues where it is deposited. Familial Amyloidic Polyneuropathy In the hereditary disease FAP, nerves and other organs are damaged by deposits of amyloid-type protein. Genetic studies have shown that the discase results from mutations in the protein transthyretin. As with the P22 tailspike protein, transthyretin contains large amounts of Besheet structure, Formation of fibrils of B-sheet structure can lead to misfolding.Alzheimer’s disease Alzheimer's disease results from aggregates of the P-amyloid peptide. Alzheimer’s afflicts 10 percent of those over 65 years old and perhaps half of those over 85. In 1991, several different research groups found that individuals with specific mutations in their amyloid precursor protein developed Alzheimer's disease as early as age 40. The body processes amyloid precursor protein into a soluble peptide known as Ab. Under certain circumstances, Ab then aggregates into long filaments that cannot be cleared by the body's usual scavenger mechanisms. These aggregates then form the B-amyloid, which make up neuritic plaque in Alzheimer patients, Aggregation and off-pathway events ‘The carly-formed intermediates are often very "sticky" (presumably ue to exposed hydrophobic surfaces) and tend to aggregate. Udgaonkar and coworkers (Nature 377, 754, 1995) recently presented evidence for the hydrophobic collapse model for barstar (a small RNase inhibitor). Sosnick et al. (Nature Structure Biology, 1, 149, 1994) re-examined the folding of cytochrome e and concluded that under certain conditions the native state was formed within 15 msec. Other studies had shown apparent intermediates on a folding pathway taking seconds. They concluded that such intermediates were artifacts, off-pathway misfolded species, and that folding involved a "funnel" with no intermediates. This is a controversial view.Non-covalent forces in proteins What holds them together? + Hydrogen bonds + Salt-bridges + Dipole-dipole interactions * Hydrophobic effect + Van der Waals forces What pulls them apart? * Conformational Entropy Protein stabilization energy is small A typical protein contains a few salt-bridges, several hundred hydrogen bonds and several thousand van der Waels interactions, In spite of all these interactions proteins are only marginally stable. Typical A G values for folding of proteins are in the range of -5 to -15 kcal/mol i.e. not much greater than the energy of 2 or 3 hydrogen bonds. This is because of several effects which cancel each other out. The enthalpy change of protein folding (A H) is dominated by hydrogen bonds. In the unfolded state the polar groups of the protein will H-bond to solvent molecules and in the folded state these polar groups will H-bond with each other. Hence the overall enthalpy change on folding is small. The hydrophobic effect is thought to make the largest contribution to A G. The hydrophobic effect attributes the poor solubility of non-polar groups in water to the ordering of the surrounding water molecules causing them to form an ice-like cluster.Dipole-Dipole Interactions Dipoles often line up in this manner. Example: a-helix Electrostatic Interactions Coulomb’s Law: V = q,q,/er Example of a hydrogen bond -N-H-O=C- Example of a Salt Bridge ‘Main Chain Main Chain cr NH & Nat HN O76 7 co al Lysine cr Glutamate va c=o0 o B o=¢ Na’ DeHydrogen bonding in waterContributions to AG 0 + Zimm-Bragg Formalism For individual biopolymers (e.g. o-helices or B-sheets) the equilibrium constant and partition function can be derived using the Zimm-Bragg formalism. The formation. of secondary structure can be divided into two steps: 1. Initiation with equilibrium constant ¢ 2. Propagation with equilibrium constant s For n residues there are n propagation steps. The overall equilibrium constant is K, = os". The free energy AG, ~-RT InK,, ~ - RTIn(o) - nRTIn(s). o os Experimental 12 1.06-1.54 Theoretical 0.1 0,002-0.008Models of protein folding * The Levinthal paradox: Two-state models + Folding pathways: Specific intermediates + The folding funnel: Multiple intermediates * Barriers in the funnel: Observable intermediates + The energy landscape: Diffusive models The Levinthal Paradox The Levinthal paradox t assumes that all of the poisible'confonnstions $= BB will be sampled with 3 equal probability until = the proper one (N = native) is found, Thus, the funnel surface looks like a hole in a golf course. The paradox <= Conformation —> states that ifa protein samples all 6% conformations it will take a time longer than the age of the universe to find the native fold (N). Note that the statistical entropy is $ = RInQ@ = R In(6") = Min(6)R in this example. M = number of residues, 6 = number of conformations per residueTwo-state folding mechanism The Levinthal view reflects the idea that protein folding would ocour as a result of a two state mechanism: USN This view suggests that the cooperativity of protein folding, i.e. the sharp transitions between unfolded and native, leads to an all-or-none mechanism. This means that there are no intermediates on the folding pathway. Because folding studies were rooted in chemistry. there has always been a strong background notion of analogy between a chemical reaction with its intermediates and transition states, However, this viewpoint suggests that the protein must hit a “hole-in-one” as indicated by Levinthal-type funnel. The Pathway Model Imagine that the a unique pathway winds through t the surface to the hole. The path starts at Aand $8 the folding goes through a unique and well-defined 4 set of conformational changes. Here the entropy | must decrease rapidly since N the number of degrees of <— Conformation —> freedom in the folding pathway is quite small compared to 6. ‘The funnel diagram is an energy diagram. The configurational entropy is implied by the width of the funnel. Ina free energy diagram the folding pathway would have a high barrier due to the large entropy change required for a unique path