Protein Denaturation Unfolded proteins or proteins in which the secondary and tertiary structure has been disrupted to create random coil are termed Denatured Denaturation can be caused by chemical means or energetic heat Albumin Denaturation Chaotropes Small molecules commonly used to denature proteins Cooperative folding Hydrophobic effect Thermal denaturation Heat Chemical denaturation with a chaotrope Protein unfolding monitored in this case by circular dichroism which measures secondary structure Abrupt two state transition suggests cooperative folding Reversible Two State Protein Folding Cooperative folding when part of the structure is disrupted it destabilizes remaining structure Denaturation can be reversible many proteins can be refolded or renatured under optimal conditions Bramley and Robson Trends Biochem Sci 1 50 1976 How Do Proteins Fold Do proteins fold through a random Levinthal s Paradox Cyrus Levinthal Back of the envelope calculations Every residue in a protein has two torsion angles sequential search pattern Assume ONLY stable conformations for each torsion angle Each residue has approximately 10 possible backbone conformations ignoring all side chains Each protein of n amino acids then has 10n possible backbone conformations Levinthal s Paradox Do proteins fold through a random sequential search pattern It takes approximately 10 13 seconds to rotate around a bond i e to sample a possible protein backbone conformation To sample all the possible conformations of all the residues in a fairly small 100 amino acid protein it would take Time to fold randomly 10n x 10 13 s 1087 s 20 billion years is 6 x 1017 s Proteins fold in seconds to milliseconds Proteins do not fold by sequential random search History Christian Anfinsen Studied the activity and stability of Ribonuclease A RNase A Protein can be isolated from cow pancreas in large quantities Protein is stable in many conditions functions in cow stomachs at very low pH Protein activity can be assayed RNA solutions are viscous RNase A cleaves bonds to make it less viscous Several disulfide bonds provide an avenue for studying partial folding of the protein Understanding of RNase A Structure Circa 1957 124 amino acid protein 4 disulfide linkages Anfinsen s Renaturation Experiments 105 7x5x3x1 possible combinations of disulfide bonds with 8 cysteines Only is right Disulfide Switching by Thiols Note Thiolate structure written as RS Reducing environment slow refolding with disulfide switching Anfinsen s renaturation experiments Allows disulfide switching Trapped in scrambled conformation Renature with preformed disulfides Anfinsen s Postulate Conclusions 1 Proteins can fold spontaneously into their native conformation under physiological conditions 2 Functional folded RNase A must be the thermodynamically favored form 3 A protein s primary sequence contains all the information necessary for a protein to adopt its native structure How Do Proteins Fold Anfinsen demonstrated proteins can refold to a favored thermodynamic state Levinthal s paradox is still in play How do proteins fold in a reasonable time frame 1 Hierarchical Protein Folding 2 Folding Funnel Hierarchical Model of Protein Folding 1 Structural nucleation similar but not precisely secondary structure based on local structural preferences 2 Local structures undergo hydrophobic collapse to molten globule state 3 Stabilization of secondary structure internal side chains pack together water is expelled from protein core Simple 2 D Folding Funnel Proteins fold via a series of conformational changes that reduce their free energy and entropy until the native state is reached There are many paths to the bottom of the funnel First step burst phase Rapid hydrophobic collapse to molten globule hydrophobic effect Small rearrangements to maximize enthalpic benefit and find native structure Proteins Can Misfold Proteins Can Misfold Alzheimer s Disease protein A Huntingtin s Disease polyQ sequences Prion diseases Scrapie in sheep Wasting Disease in deer Mad Cow Disease and Creutzfeldt Jakob disease CJD in people In all of these brain tissue gains spongelike appearance due to neural degeneration Misfolded prion protein appears to be responsible for this Disease develops over the course of many years Proteins Can Misfold for cellular prion protein not infectious Prion proteins PrPC PrPSc for scrapie prion protein harmful and infectious PrPSc catalyzes conversion of PrPC to harmful PrPSc form Structure of PrPC protein No detectable structure in residues Model structure of PrPSc protein more sheet Amyloids Model of amyloid fiber Amyloid deposits large red spots and intracellular tangles dark Alzheimer s disease Alzheimer s Disease Alzheimer s disease is also characterized by amyloid plaques is very complex Formation of the fibrils and tangles in Alzheimer s disease The damage is currently thought to be caused by small oligomers not the deposited plaques Mutations that destabilize proteins involved in formation of amyloid tangles can lead to early onset Alzheimer s disease Molecular Chaperones Molecular chaperones function to inhibit inappropriate interactions between potentially complementary surfaces and disrupt unsuitable liaisons so as to facilitate more favorable associations More simply Chaperones help to minimize aggregation and provide a framework for proper protein folding Chaperonin proteins aid in the proper folding of misfolded proteins with exposed hydrophobic patches Classic example is the large multi subunit protein complex formed from GroEL and GroES in E coli Chaperonin Protein Family Extra GroEL GroES are very well studied chaperonins Active complex formed from 14 GroEL subunits along with a GroES cap Surface view Sliced in half GroEL GroES Activity Extra When no GroES cap in place Hydrophobic patches of A domains bind hydrophobic patches of misfolded proteins GroES binds and ATP hydrolysis causes rotation of A domain Trapped and isolated protein is surrounded by polar environment for refolding Conformational flexibility can be important Some sequences can take on multiple structures Some proteins are natively unfolded Some proteins required processing cleaving bonds post translational modification lipidation glycosylation etc to become functional Some modifications induce structural changes Questions Problems 34 35 36 40 41 42 43 45 46 Know All material from this section Do not need to memorize names of proteins used as illustrations for which protein