1Protein Structure – FoldingDr. Leisha Mullins20132Protein Folding and Stability•The primary sequence contains all the information a protein needs to adopt its tertiary and quaternary structure or “native structure”•Native structure is generally only slightly more stable than the unfolded structure– 0.4 kJ /mole/amino acid•Weak non-covalent interactions along with covalent crosslinks (e.g. disulfide bonds) stabilize the native structure•But how does fold from a linear chain to native structure?smaller protein= more disulfide bonds3Denaturation•It is generally easier to study how a protein unfolds or denatures•Loss of structure results in lose of function – There is only a single native structure – but there is not “one” denatured structure– Rather tend to call the denatured state a “random” coil•Many proteins exhibit a two state folding process (i.e. either folded or unfolded)reversible50% unfolded means that 50% of the proteins in solution still have native structure and 50% are unfolded4Denaturants• Non-covalent interactions are weak• Disruptions cause proteins to denature–Heat –pH – Detergents •SDS –Chaotropicagents•Urea, guanidniumchloridedisrupts ionic interactions & H bondssolubilize hydrophobic groups (increase solubility) Identify and tell what they will affect5Oxidation / Reductions •Remember chemical crosslinks like disulfide bonds also stabilize or constrain the structure of a protein•Reduce with chemicals like DTT and βmercaptoethanol and enzymesblock after reductionChristian Anfinsin and Ribonuclease A• In 1957, showed that ribonuclease A could be denatured –unfold to a “random” coil • AND that it could spontaneously renature–refold to the native tertiary structure 6ribonuclease slow folding intermediates have incorrect disulfide bonds7Protein Folding PathwaysWhat are the driving forces behind protein folding?•LeventhialParadox –if a polypeptide chain searched all possible conformations on its path to it native tertiary structure it would take longer than the apparent age of the universe ( 4 x 109years or 4 billion years) to fold•Proteins can fold on a millisecond time scale•So how do they get from the primary sequence to the tertiary and quaternary structure?proteins have shortcuts8Protein Folding •Secondary Structures probably form first•Followed closely by a hydrophobic collapse•Final steps involve the formation of long rand interactions between secondary•Folding may involve intermediate states known as molten globulescollapse into middle of protein9Thermodynamics of Protein Folding• Anfinsen’s experiments showed that protein folding is a physical process that depends on the amino acid sequence and the solvent•This implies that folding can be understood from a thermodynamic perspective–The folded or native structure should have the global minimum free energybc delta G is less than 0 (folds spontaneously); must go from low energy to high energy10Thermodynamics of Protein Folding • ∆G < 0 for folding•Unfolded polypeptide- high entropy-high energy•Native conformation - single low entropy-low energyspontaneousbc unfolded has more disorderentropymore orderenergythe smoother the funnel, the fewer detectable intermediates11Favorable and Unfavorable Thermodynamic StatesUnfavorable•Conformation entropy•The folding of a polypeptide chain to a single folded state is entropically disfavored∆S < 0Favorable • Internal Interactions• enthalpically favorable ∆H < 0• Hydrophobic effect• entropically favorable ∆S > 0∆G = ∆H -T∆SFor protein folding ∆G < 0 bury all hydrophobic groupstemperature determines whether protein is folded or unfolded12Unfolding and Temperature•Proteins will begin to unfold when ∆G =0•Therefore, ∆H =T∆S•This temperature is known as the denaturation temperature•Belowdenat. temp – protein folded•Abovedenat. temp – protein unfoldedfolded unfolded13Protein Dynamics•Proteins are NOT “frozen” in solution•Rather they are “breathing”•There can be local fluctuations vs global unfolding•Studies of local fluctuations tend to show– Peptide backbone areas not in stable secondary structures show higher fluctuations– However, with in secondary structural elements there is a varying degree of fluctuations•Alpha helices tend to “fray” at the termini •Beta sheets are more stable in the center– Possibly the most stable areas of structure are the same areas that act as the folding nucleation sitesstable structure= alpha helix or beta sheetTable 6-2 p173•Interconversion of cis and trans Proline•Peptide prolyl cis-trans isomerase (PPI)proline= cis 10%helps with isomerization of Proline15Protein Folding in vivoProtein Folding conditions in vivo are slightly different than in vitro folding conditions•The redox environment in vivo can be different from in vitro conditions– In cells, sometimes the wrong disulfide bonds are formed– The enzyme protein disulfide isomerase (PDI) is used in cells to reduce and reoxidize disulfide bondsinside cellhelps break incorrect bond allowing the protein to fold correctly16Molecular chaperons•Cellular concentration of proteins can be very high leading to aggregation of partially folded protein intermediates– Molecular chaperons bind unfolded and partially folded polypeptide chains sequestering them in an environment that the favorable for protein folding– Molecular chaperons can also capture misfolded proteins and facilitate the correct folding – Two classes have been well characterized•Hsp70 family•chaperoninsprotein folds insideknow why they are in the cell and what they do17Protein Folding Diseasesassociated with incorrect foldingdisease states18Protein Folding Diseases19Protein Folding DiseasesDisease Affected protein Mechanism Sickle Cell anemia hemoglobin deformation of erythrocytes Alzheimer ββββ amyloid peptide Plaques form in neural tissue Creutzfeldt – Jacob Prion Plaques form in neural tissue Hereditary emphasema αααα1-antitrypsin slow folding allows target, elastase, to destroy lung tissue Cystic fibrosis CFTR (cystic fibrosis transmembrane conductance regulator) Folding intermediates do not dissociate from chaperones preventing protein from reaching target membrane aggregationproteinion channel: as ion goes out, water goes out to lubricate lungs; without protein, lungs cannot be lubricatedknow names and that