Continue portion of Protein

1. Continue portion of Protein

2. The biological function of protein is due to the three-dimensional structure which decided by the primary structure. • Proteins are at the center of action in biological processes.

3. §3.1 The relationship between primary structure and biological function • The potential function of a protein is specified by its primary structure

4. Proteins having similar amino acid sequences demonstrate the functional similarity. • The alternation of key AAs in a protein will cause the lose of its biological functions.

5. Sequences of Cytochrome C • Cytochrome C is a protein which can be found in all aerobic organisms.

6. Proteins having similar amino acid sequences demonstrate the functional similarity. • The alternation of key AAs in a protein will cause the lose of its biological functions.

7. — Sickle cell anemia

8. Hemoglobin

10. Sickle cell anemia is caused by a single amino acid replacement on the β subunit: Glu6→Val6.

11. §3.2 The relationship between function and conformation

12. 1. Proteins of incorrect structures have no proper biological functions, even their amino acid sequences are remained in a right order. 2. A particular spatial structure of a protein is strongly correlated with its specific biological functions.

13. Bovine nuclease • 124 AAs, 4 disulfide bonds (105 possibilities)

14. Urea, β - mercaptoethanol Native ribonuclease Removal of denaturantAnd slow oxidation Denatured ribonuclease active Inactive

15. The denatured protein remains its primary structure, but no biological function. The renatured protein will restore its functions partially or fully depending upon the correctness of the refolded structure. • Only the correct form has the enzymatic activity.

16. Proteins of incorrect structures have no proper biological functions, even their amino acid sequences are remained in a right order. • A particular spatial structure of a protein is strongly correlated with its specific biological functions.

17. Mb, Hb share a similar tertiary structurealthough their sequence identity is only about 18%. Each peptide includes a heme prosthetic group. They all can combine with oxygen molecules. Hemoglobin

18. Concerted versus sequential low O2 affinity T-state and high affinity R-state.

19. The O2–binding to the 1st subunit enhances the O2–binding to the 2nd and 3rd subunits. Such process further enhances the O2–binding to the 4th subunit significantly.

20. The quaternary structure of Hb changes markedly for the tense (T) form to the relaxed (R) form upon oxygenation.

21. Kinetic curve:O2 dissociation curve sigmoidal curve With the increase of oxygen partial pressure, hemoglobin and oxygen binding capacity increased. Hb binds O2 in a positive cooperative manner, which enhances the O2 transport. • Carry O2 in the blood from lung (high O2 partial pressure, combine)to the other tissue (low O2 partial pressure, release )in the body

22. Kinetic curve:O2 dissociation curve hyperbolic curve sigmoidal curve Low oxygen pressure, Mb will be able to quickly combine with oxygen and reached saturation (not easy to dissociate unless under very low oxygen pressure) • Mb has a higher affinity for O2 than Hb (higher P50).

23. Function: Hb: Carry O2 in the blood from lung to the other tissue in the body. Mb: store the O2 in the tissue.

24. Allosteric effect • The behavior that the ligand-binding to one subunit causes structural changes and stimulate the further binding to other subunits is termed as allosteric effect. • The protein is allosteric protein, and the substrate is allosteric effector. • Allosteric effect can be influenced by activators as well as inhibitors.

25. Section 4 Protein Physical-Chemical Properties and Purification

26. §4.1 the characters of protein §4.1.1 Amphoteric Isoelectric point • AAs in solution at certain pH are predominantly in dipolar form, fully ionized but without net charge due to -COO- and -NH3+ groups. • This characteristic pH is called isoelectric point, designated as pI. • pI is determined by pK, the ionization constant of the ionizable groups.

27. +H+ +OH- +H+ +OH- pH>pI pH<pI pH=pI amphoteric cation anion

29. Side-chains of a protein have many ionizable groups, making the protein either positively or negatively charged in response to the pH of the solution. • The pH at which the protein has zero net-charge is referred to as isoelectric point (pI).

30. pI of most protein is ~5.0, and negatively charges in body fluid (pH7.4) • pI > 7.4: basic proteins: protamine, histone • pI < 7.4: acidic proteins: pepsin

31. §4.1.2 Colloid property • Diameter: 1~100nm, in the range of colloid; • Hydrophilic groups on the surface form a hydration shell; • Hydration shell and electric repulsion make proteins stable in solution.

32. + － acid base － + + － － base acid + + － － － + + － isoelectric point (hydrophilic) + － + + － － + base acid + － － + － + + － － Precipitation of protein colloid positively charged (hydrophilic) negatively charged (hydrophilic) dehydration dehydration dehydration positively charged (hydrophobic) Instable protein (deposition) negatively charged (hydrophobic)

33. The denatured proteins tend to - decrease in solubility; - increase the viscosity; - lose the biological activity; - lose crystalizability; - be susceptible to enzymatic digestion. §4.1.3 Protein denaturation The process in which a protein loses its native conformation under the treatment of denaturants is referred to as protein denaturation.

34. Cause of denaturation the disruption of hydration shell and electric repulsion Denaturants physical: heat, ultraviolet light, violent shaking, … chemical: strong acids, bases, organic solvents, detergents, … Applications sterilization, lyophilization

36. Renaturation • Once the denaturants are removed, the denatured proteins tend to fold back to their native conformations partially or fully. • The renatured proteins can restore their biological functions.

37. Renaturation

38. Protein precipitation The denatured proteins expose their side chains or the inner part to the aqueous environment, which causes the proteins aggregated and separated out from the aqueous solution.

39. Protein coagulation • When the denatured proteins become insoluble fluffy materials, heating denatured proteins will turn them into a hard solid which are not soluble even strong acids and bases are applied. • Coagulation is an irreversible process.

40. §4.1.4 UV absorption • Trp, Tyr, and Phe have aromatic groups of resonance double bonds. • AAs have a strong absorption at 280nm. • Both free and incorporated AAs show this absorption.

42. §4.1.5 Coloring reactions • Biuret reaction: peptide bonds and Cu2+ under the heating condition to form red or purple chelates. • Used for determine the hydrolysis of proteins since free amino acids do not react. • Amino acids can react with ninhydrin to form a chemicals having maximal adsorption at 570 nm. • Used for quantifying the free amino acids.

43. §4.2 Isolation and purification • Homogenization and centrifugation • Dialysis • Precipitation • Chromatography • Electrophoresis

44. §6.1.a Homogenization • Rupture the plasma membrane to release the intracellular components into the buffered solution • Sonication, French pressure, mechanical grinding, • Chemical reagents, lysozymes

45. Centrifugation • Because of the differences in size and shape, proteins will sediment gradually under the centrifugal force until the sedimentation force and buoyant force reach the balance. • The sedimentation behavior is described in sedimentation coefficient (S) which is proportional to the molecular weight.

46. Differential centrifugation

47. Differential centrifugation

48. Rate-zone centrifugation

49. §6.1.b Dialysis • Proteins, as macromolecules, cannot pass through the semipermeable membrane containing pores of smaller than protein dimension, thus large proteins and small molecules can be separated. • Dialysis can be used for protein purification, desalting, and condensation.