Protein Structure Folding Dr Leisha Mullins 2013 1 Protein 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 bonds 2 It is generally easier to study how a protein unfolds or denatures Loss of structure results in lose of function revers ible Denaturation 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 50 unfolded means that 50 of the proteins in solution still have native structure and 50 are unfolded 3 Denaturants Non covalent interactions are weak Disruptions cause proteins to denature Heat pH Detergents disrupts ionic interactions H bonds solubilize hydrophobic groups increase solubility SDS Chaotropic agents Urea guanidnium chloride 4 Identify and tell what they will affect Oxidation Reductions Remember chemical crosslinks like disulfide bonds also stabilize or constrain the structure of a protein Reduce with chemicals like DTT and mercaptoethanol and enzymes block after reduction 5 Christian Anfinsin and Ribonuclease A In 1957 showed that ribonuclease A could be denatured AND that it could spontaneously renature refold to the native tertiary structure 6 ribonuclease slow folding intermediates have incorrect disulfide bonds unfold to a random coil Protein Folding Pathways What are the driving forces behind protein folding Leventhial Paradox 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 109 years 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 shortcuts 7 Protein 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 globules collapse into middle of protein 8 Thermodynamics 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 energy bc delta G is less than 0 folds spontaneously must go from low energy to high energy 9 Thermodynamics of Protein Folding entro py G 0 for folding spontaneo us Unfolded polypeptide high entropy high energy Native conformation single low entropy low energy ener gy bc unfolded has more disorder more order 10 the smoother the funnel the fewer detectable intermediates Favorable and Unfavorable Thermodynamic States G H T S For protein folding G 0 Favorable Internal Interactions enthalpically favorable H 0 Hydrophobic effect entropically favorable S 0 bury all hydrophobic groups Unfavorable Conformation entropy The folding of a polypeptide chain to a single folded state is entropically disfavored S 0 11 temperature determines whether protein is folded or unfolded Unfolding and Temperature Proteins will begin to unfold when G 0 Therefore H T S This temperature is known as the denaturation temperature Below denat temp protein folded Above denat temp protein unfolded folded unfolded 12 Protein 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 stable structure alpha helix or beta sheet 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 sites 13 proline cis 10 helps with isomerization of Proline Interconversion of cis and trans Proline Peptide prolyl cis trans isomerase PPI Table 6 2 p173 inside cell Protein Folding in vivo Protein 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 bonds helps break incorrect bond allowing the protein to fold correctly 15 protein folds inside Molecular 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 know why they are in the cell and what they do Hsp70 family chaperonins 16 17 disease states associated with incorrect folding Protein Folding Diseases Protein Folding Diseases 18 know names and that they are folding diseases Protein Folding Diseases Disease Affected protein Mechanism Sickle Cell anemia hemoglobin deformation of erythrocytes Alzheimer amyloid peptide Plaques form in neural tissue Creutzfeldt Jacob Prion Plaques form in neural tissue Hereditary emphasema 1 antitrypsin slow folding allows target elastase to destroy lung tissue Cystic fibrosis CFTR aggregat ion prot ein cystic fibrosis transmembrane conductance regulator Folding intermediates do not dissociate from chaperones preventing protein from reaching target membrane ion channel as ion goes out water goes out to lubricate lungs without protein lungs cannot be lubricated 19 Prion Diseases infectious agent consisting solely of proteins was first proposed in the early 60s Stanley Prusiner first coined the term proteinaceous infectious particle PRION in the early 80s Received the Nobel Prize in Medicine in 1992 According to the CDC the