Chapter 5: Outline. Protein Function. Proteins by Shape-2. Proteins by Shape-1. Proteins by Composition

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1 hapter 5: utline Amino Acids Amino acid classes Bioactive AA Modified AA Peptides Proteins (We are here) Protein structure Fibrous proteins Globular proteins tereoisomers Titration of AA AA reactions 5P2-1 Protein Function 1. atalysis 2. tructure 3. Movement 4. Defense 5. Regulation 6. Transport 7. torage 8. tress Response 5P2-2 Proteins by hape-1 Fibrous proteins exist as long stranded molecules: Eg. ilk, collagen, wool. A collagen segment in space-filling mode illustrates this point. Red spheres represent oxygen, grey carbon, and blue nitrogen Proteins by hape-2 Globular proteins have somewhat spherical shapes. Most enzymes are globular. Eg. myoglobin, hemoglobin. Myoglobin in space-filling mode is the chosen example. 5P2-3 5P2-4 Proteins by omposition imple ontain only amino acids onjugated simple protein (apoprotein) prostetic group (nonprotein) glycoproteins lipoproteins metaloproteins etc. oloprotein 5P2-5 Four Levels of Protein tructure Primary, 1 o the amino acid sequence econdary, 2 o 3-D arrangement of backbone atoms in space Tertiary, 3 o 3-D arrangement of all the atoms in space Quaternary, 4 o 3-D arrangement of subunit chains 5P2-6 1

2 Determining Primary tructure 1. ydrolyze protein with hot 6M l. Identify AA and % of each. Usually done by chromatography 2. Identify the -term and -term AAs -term via carboxypeptidase -term via anger s Reagent, DFB 2,4-dinitrofluorobenzene ften step 2 can be skipped today. Det. Primary tructure: 2 3. electively fragment large proteins into smaller ones. Eg. Tripsin: cleave to leave Arg or Lys as -term AA Eg. hymotrypsin: cleave to leave Tyr or Trp or Phe as -term AA Eg. yanogen bromide cleaves at internal Met leaving Met as -term homoserine lactone 5P2-7 5P2-8 Det. Primary tructure: 3 4. Determine AA sequence of peptides with AA sequencer using Edman s reagent: phenyl isothiocyanate which reacts with the -term AA ee the next slide 5P2-9 Det. Primary tructure: 3b R1 R1 protein Edman s reagent 3 aqueous acid RAR Thiazoline derivative Phenylthiohydantoin (PT) derivative of -term AA 5P2-10 Det. Primary tructure: 4 5. Reassemble peptide fragments from step 3 to give protein. An example follows on the next slide. Det. Primary tructure: 4b A twelve AA peptide was hydrolyzed. Trypsin hydrolysis: Leu-er-Tyr-Gly-Ile-Arg Thr-Ala-Met-Phe-Val-Lys hymotrypsin hydrolysis ne is -term Val-Lys-Leu-er-Tyr Lys is internal! Gly-Ile-Arg Thr-Ala-Met-Phe Deduce the AA sequence 5P2-11 5P2-12 2

3 Det. Primary tructure: 4c Keeping in mind the -term AA and overlaping the sequences properly gives: Tr Leu-er-Tyr-Gly-Ile-Arg t Gly-Ile-Arg t Val-Lys-Leu-er-Tyr Tr Thr-Ala-Met-Phe-Val-Lys t Thr-Ala-Met-Phe The complete sequence is: Thr-Ala-Met-Phe-Val-Lys-Leu-er-Tyr-Gly-Ile-Arg 5P2-13 econdary tructure The two very important secondary structures of proteins are: α-helix β-pleated sheet Both depend on hydrogen bonding between the amide and the carbonyl further down the chain or on a parallel chain. 5P2-14 α elix: Peptide w bonds β heet: stick form Protein G First six = to hydrogen bonds shown bonds in dotted red-blue bonds shown in dotted red-blue hain segment 1 eg 2 hain 1 eg 3 eg 4 5P2-15 5P2-16 B heet: Lewis tructure -term -term -term -term Antiparallel sheet -term -term -term -term Parallel sheet 5P2-17 upersecondary tructure Reverse turns in a protein chain allow helices and sheets to align side-by-side ommon AA found at turns are: glycine: small size allows a turn proline: geometry favors a turn 5P2-18 3

4 upersecondary tructure: 2 ombinations of α helix and β sheet. Tertiary tructure The configuration of all the atoms in the protein chain: side chains prosthetic groups helical and pleated sheet regions βαβ αα β meander 5P2-19 5P2-20 Tertiary tructure: 2 Protein folding attractions: 1. oncovalent forces a. Inter and intrachain bonding b. ydrophobic interactions c. Electrostatic attractions to - ionic attraction d. omplexation with metal ions e. Ion-dipole 2. ovalent disulfide bridges 5P2-21 Tertiary interactions: diag. disulfide 2 2 metal coord n 3 ionic Mg 2 Polypeptide hain Ion-dipole hydrophobic 3 bonds or dipole 5P2-22 Domains Domains are common structural units within the protein that bind an ion or small molecule. Quaternary tructure-1 Quaternary structure is the result of noncovalent interactions between two or more protein chains. ligomers are multisubunit proteins with all or some identical subunits. The subunits are called protomers. two subunits are called dimers four subunits are called tetramers 5P2-23 5P2-24 4

5 Quaternary tructure-2 If a change in structure on one chain causes changes in structure at another site, the protein is said to be allosteric. Many enzymes exhibit allosteric control features. emoglobin is a classic example of an allosteric protein. 5P2-25 Denaturation -loss of protein structure, 2 o 4 o, but not 1 o. 1. trong acid or base 2. rganic solvents 3. Detergents 4. Reducing agents 5. alt concentrations 6. eavy metal ions 7. Temperature changes 8. Mechanical stress 5P2-26 Denaturation-2 Denaturing destroys the physiological function of the protein. Function may be restored if the correct conditions for the protein function are restored. But! ooling a hardboiled egg does not restore protein function!! Fibrous Proteins Fibrous proteins have a high concentration of α-helix or β-sheet. Most are structural proteins. Examples include: a-keratin collagen silk fibroin 5P2-27 5P2-28 Globular Proteins Usually bind substrates within a hydrophobic cleft in the structure. Myoglobin and hemoglobin are typical examples of globular proteins. Both are hemoproteins and each is involved in oxygen metabolism. Myoglobin: 2 o and 3 o aspects Globular myoglobin has 153 AA arranged in eight α-helical regions labeled A-. The prosthetic heme group is necessary for its function, oxygen storage in mammalian muscle tissue. is E7 and F8 are important for locating the heme group within the protein and for binding oxygen. A representation of myoglobin follows with the helical regions shown as ribbons. 5P2-29 5P2-30 5

6 Myoglobin: 2 o and 3 o aspects ome helical regions eme group with iron (orange) at the center 5P2-31 The eme Group - 2 of is F8 binds to 3 fifth site on 3 the iron. Fe(II) 2 Pyrrole ring 3 is E7 acts as a gate for oxygen P2-32 Binding ite for eme Lower is bonds covalently to iron(ii) xygen coordinates to sixth site on iron and the upper is acts as a gate for the oxygen. Fe 5P2-33 emoglobin A tetrameric protein two α-chains (141 AA) two β-chains (146 AA) four heme units, one in each chain xygen binds to heme in hemoglobin cooperatively: as one 2 is bound, it becomes easier for the next to bind. Lengthy segments of the α and β chains homologous to myoglobin. 5P2-34 emoglobin: ribbons hemes Each chain is in ribbon form and color coded. The heme groups are in space filling form xygen Binding urves xygen bonds differently to hemoglobin and myoglobin. Myoglobin shows normal behavior while hemoglobinn shows cooperative behavior. Each oxygen added to a heme makes additon of the next one easier. The myoglobin curve is hyperbolic. The hemoglobin curve is sigmoidal. 5P2-35 5P2-36 6

7 xygen Binding urves-2 The Bohr Effect ( and b) Lungs: p higher than in actively metabolizing tissue. (Low ). b binds oxygen and releases. Muscle at Work: p lower ( product of metabolism). b releases oxygen and binds. 5P2-37 b 2 2 metabolism 2 - b- 2 in lungs 5P2-38 7