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Lecture 6

BIOC12Fall2012 Lecture Week 6

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Department
Biological Sciences
Course
BIOC12H3
Professor
Rongmin Zhao
Semester
Fall

Description
BIOC12FALL2012 Lecture Week 6: Protein Folding Structure and Function are not Always Linked  Because structure depends on sequence, and because function depends on structure, it is tempting to imagine that all proteins of similar structure should share a common function, but this is not always true  Some proteins of similar domain structure have different functions  domain has its own function even when cleaved from the larger protein  Motif does not form a globular protein and does not have a function away from the other parts  Some proteins of similar function possess very different structures   in a, all 3 proteins have a barrel structure, but they serve different functions  in b, the 2 proteins serve the same function but have different strucutres Denaturation Leads to Loss of Protein Structure and Function  the proteins of egg are denatured during cooking  ovalbumin structure is modified, so no longer holds the same function Protein is not stable and can be easily denatured  note: the amount of energy needed for denaturation is low  destabilization of just a few weak interactions leads to complete loss of native conformation  factors affecting protein stability (environmental factors): 1. temperature 2. pH: at pH other than optimal, changes the protonation or deprotonation states of ionic groups, disrupting hydrophobic interactions 3. detergents: disrupt interactions and destabilize the protein 4. chemicals: chaotropic agents  ions or small organic molecules that increase the solubility of non polar substrates in water (usually used at 5-10M concentration) Chaotropic agents 1  denature proteins into random coils by allowing water molecules to solvate non polar goups in the interior of the proteins  Protein Denaturation  denaturation = conversion from native conformation with loss of biological activity  native conformation (N) usually refers to the naturally occurred active status of a protein  after denaturation, some proteins may completely unfold and form a random coil  totally disorded and unfolded (U) structure, while other retain some internal structures Denaturation Leads to Loss of Protein Structure and Function  proteins can be denatured by heat, with commensurate loss of function  Measure the denaturation of proteins  monitored by changes in:  UV absorption (blue): once denatured, you will have more amino acids exposed, therefore the concentration/absorption of aromatic amino acids will increase compared to the original sample where the protein is in its native conformation  Viscosity (red): once denatured, the proteins would be made into random coils, which are longer and more linear molecules than globular  this increases intermolecular interaction and therefore increases viscosity  Optical density (green): increases as protein becomes denatured  Indirect enzymatic assay  Melting Temperature (TM): the temperature at which half of the molecules are in denatured form  this notion is shared among proteins and nucleic acids  Below is a thermal denaturation curve of RNAse A  the transition from N to U is very sharp 2  Sharp Transition – Cooperative denaturation  a sharp transition from N to U indicates a cooperative denaturation  the whole structure is denatured simultaneously (S-shaped) Protein Denaturation and Renaturation: example of RNAse A  experiment done by Christian Anfinsen in 1957  RNAse A is made up of 124 aa  endoribonuclese that specifically degrades single stranded RNA at C and U residues; the protein contains 4 disulfide bonds  Denaturation: 8M urea and b-mercaptoethanol Experimental Details  Treatment with an 8M urea solution containitnng b-merc completely denatures most proteins  The urea breaks intramolecular H bonds, and the mercaptoethanol reduces disulfide bridges to sulhydryl groups (SH)  When these checmicals are removed by dialysis, the –SH groups on the unfolded chain oxidize spontaneously to re-form disulfide bridges and the polypeptide chain simultaneously refolds into its native conformation Anfinsen’s Classic Protein Folding Experiment  Ribonuclease can be unfolded by treatment with urea, and b-mercaptoethanol (MCE) cleaves disulfide bonds  Anfinsen showed that ribonuclease structure and function could be restored under appropriate conditions  Structure is first different (Scrambled) but upon the addition of a little MCE and heat the right disulfide bonds are reformed and the protein goes back to its original form 3  Conclusion of Anfinsen RNase denaturation and renaturation experiment  3D structure is determined by the primary structure or  the protein sequence contains all information about the protein  sequence determines structure Protein folding is not the result of a random conformational search  the protein renaturation is very fast, usually within the time of seconds  proteins cannot fold by sampling all possible conformations  this implies that proteins actually fold via specific folding pathways  peptide bonds in a protein = number of amino acids - 1 Postulated themes of protein folding Postulated Themes of Protein Folding Random coil < 5ms molten globule state (collapse sate) Local folding to form secondary structure <5-1000ms Stabilizing secondary structure
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