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Huge number of possibilities Classified in many ways: solubility composition shape physical properties function 3-D structure Solubility : Solubility Albumins Soluble in water and salt soln’s Globulins Sparingly soluble in water but soluble in salt solutions Prolamines Soluble in 70-80% EtOH but insol in water and absolute EtOH Histones Soluble in salt soln’s Scleroproteins Insoluble in water or salt soln’s CompositionSimple vs. Conjugated : CompositionSimple vs. Conjugated Simple- Conjugated- Apoprotein- Holoprotein- Prosthetic group- Shape: Globular vs. Fibrous : Shape: Globular vs. Fibrous Rasmol Demo 10:1 ratio arbitrary division Physical Properties : Physical Properties Not discussed Function : Function Enzymatic catalysts- next chapter Transport and storage of molecules- Hb, ferritin Mechanical functions- elastin Movement- myosin Protection- Ab Information processing- rhodopsin Regulatory- renin Other Structure : Structure Primary (1o)- sequence of amino acids Secondary (2o)- local 3-D shape a-helix ß-sheet collagen triple helix Tertiary (3o)- global 3-D shape Quaternary (4o)- relation of polypeptides 1o Structure : 1o Structure 1o Structure- sequence of amino acids (disulfide bond locations) MUST have pure protein Protein Purification Starting Material : Protein Purification Starting Material Start with a source very rich in protein: Organism, tissue, cell type Can you isolate a particular organelle as a starting purification step? Protein Purification Salting-in/salting out : Protein Purification Salting-in/salting out Process Bigger salts work better: (NH4)2SO4 Bigger proteins ppt at lower [salt] Dialysis Protein Purification Size exclusion chromatography : Protein Purification Size exclusion chromatography Separate by size (number of amino acids) Protein Purification Ion-exchange chromatography : Protein Purification Ion-exchange chromatography Separate on basis of protein charge lys/arg vs asp/glu net charge Positively charged beads Negatively charged beads Protein Purification Affinity chromatography : Protein Purification Affinity chromatography Takes advantage of a specific binding property of the protein. Protein Purification Electrophoresis : Protein Purification Electrophoresis Preparative electrophoresis can be used, but analytical electrophoresis used more often to see how pure the protein is. Protein Amino Acid Composition : Protein Amino Acid Composition Acid hydrolysis- destroys Ser, others Base hydrolysis- destroys Gln, others Protein Sequencing : Protein Sequencing 1. What is amino acid composition? 2. What is amino terminus? Sanger’s reagent 3. What is carboxy terminus? Limited carboxypeptidase digestion 4. What is sequence? Edman degradation Protein Sequencing Continued : Protein Sequencing Continued 5. Fragment protein cyanogen bromide proteases 6. Align fragments Protein Sequencing : Protein Sequencing 1. What is amino acid compostion? 2. What is amino terminus? -Sangers reagent 3. What is carboxy terminus? -Carboxypeptidase 4. What is sequence? -Edman degradation 5. Fragment protein cyanogen bromide proteases 6. Align fragments Protein Sequencing Example : Protein Sequencing Example 1. Isolated pure protein 2. What is amino acid composition? Acid hydrolyze, 2D chromatography and detect Gly- 2 Val- 2 Pro- 2 Ser- 2 His- 1 Phe- 1 Tyr- 1 Trp- 1 Met- 1 Arg- 3 Lys- 3 Glx- 1 Protein Sequencing Example : Protein Sequencing Example 3. What is amino terminus?: Serine Sangers reagent Protein Sequencing Example : Protein Sequencing Example 4. What is carboxy terminus?: Valine Carboxypeptidase Limited digestion (short time, low temp) gives a single major a.a. Protein Sequencing Example : Protein Sequencing Example 5. What is sequence? Edman degradation Protein Sequencing Example : Protein Sequencing Example 6. Fragment protein cyanogen bromide: 2 pieces Chymotrypsin: 5 pieces Trypsin: ? Pieces Data on board Protein Sequencing Example : Protein Sequencing Example 7. Align fragments Ser-... Much Data, Much Work : Much Data, Much Work The Process Just Described is a Huge Amount of Work : The Process Just Described is a Huge Amount of Work Method of choice today is to sequence DNA: drawback Only with prior knowledge do we move forward Forces Involved in 1o Structure : Forces Involved in 1o Structure Strong peptide bond disulfide bond Primary Structure Determines Secondary Structure : Primary Structure Determines Secondary Structure Dipeptide model- not all conformations are possible Ramachandran plot Secondary (2o)- local 3-D shape a-helix ß-sheet collagen triple helix a-helix : a-helix Compact pitch: rise/residue know the dimensions location of R groups every fourth amino acid R group interacts amino acids NOT Pro forces responsible h-bonds parallel to axis ß-sheet : ß-sheet More extended conformation location of R groups alternate amino acids “all” forces involved h bonds perpendicular Parallel vs. Antiparallel Usually short collagen triple helix : collagen triple helix Kinky Pro-X-Gly or Hyp-X-Gly Pro makes kinks X varies Why Gly? Forces Involved in 2o Structure : Forces Involved in 2o Structure Weak hydrogen bonds electrostatic interactions metal ion coordination hydrophobic effect 3o Structure : 3o Structure 3o Structure- Global 3-D shape How are the 2o structures arranged in relation to each other? Predicting 3o Structure : Predicting 3o Structure Shape of 6,000 proteins determined by X-ray crystallography More than 500,000 sequenced Computers allow prediction if sequence known 3o Structure : 3o Structure Huge number of possible structures Generalizations interior hydrophobic, exterior hydrophillic form follows function: motifs EX: helix-loop-helix motif EX: beta bend motif EX: Greek Key motif EX: ß-a-ß motif Protein Folding : Protein Folding Not just any old way For a given protein, all molecules have the same shape. folding occurs in stages Domains-several motifs usually combine to form compact globular structures Forces Involved in 3o Structure : Forces Involved in 3o Structure Weak hydrogen bonds electrostatic interactions metal ion coordination hydrophobic effect Denaturation : Denaturation Denaturation- disruption of the normal 3D shape agents: alcohol weak acid or base heat detergents reducing agents 4o Structure : 4o Structure 2 or more subunits arranged in relation to each other held together by noncovalent interactions 2 or more subunits ---> dimer, trimer, etc. homodimer vs. heterotrimer, etc. Forces Involved in 4o Structure : Forces Involved in 4o Structure Weak hydrogen bonds electrostatic interactions metal ion coordination hydrophobic effect You do not have the permission to view this presentation. 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