Membrane Transport; Nucleic Acid Chemistry

1. Membrane Transport;Nucleic Acid Chemistry Andy HowardBiochemistry Lectures, Spring 2019Tuesday 5 March 2019

2. Membrane transport;Nucleic Acid Chemistry • Proteins facilitate transport in several ways • Nucleotides are phosphate esters of nucleosides, which in turn are riboglycosides of nucleic acid bases Nucleic Acid Fundamentals

3. Active transport Moving big objects Nucleic acid bases Nucleosides & -tides Deoxynucleosides Nucleic Acid Chemistry DNA structure Low-level High-level What we’ll discuss Nucleic Acid Fundamentals

4. Primary active transport • Energy source is usually ATP or light • Energy source directly contributes to overcoming concentration gradient • Bacteriorhodopsin: light energy used to drive protons against concentration and charge gradient to enable ATP production • P-glycoprotein: ATP-driven active transport of many nasties out of the cell Nucleic Acid Fundamentals

5. Secondary active transport • Active transport of one solute is coupled to passive transport of another • Net energetics is (just barely) favorable • Generally involves antiport • Bacterial lactose influx driven by proton efflux • Sodium gradient often used in animals Nucleic Acid Fundamentals

6. Complex case: Na+/K+ pump • Typically [Kin] = 140mM,[Kout] = 5mM,[Nain] = 10 mM, [Naout] = 145mM. • ATP-driven transporter:3 Na+ out for 2 K+ inper molecule of ATP hydrolyzed Diagram courtesy Steve Cook Nucleic Acid Fundamentals

7. Quantitation for Na+/K+ pump • 3Na+ out: 3*6.9 kJmol-1,2K+ in: 2*8.6 kJmol-1= 37.9 kJ mol-1 needed • ~ one ATP Sus Na/K pump321kDa heterooligomerEC 3.6.3.9PDB 3WGU, 2.8Å Nucleic Acid Fundamentals

8. How do we transport big molecules? • Proteins and other big molecules often internalized or secreted by endocytosis or exocytosis • Special types of lipid vesicles created for transport Nucleic Acid Fundamentals

9. Receptor-mediated endocytosis • Bind macromolecule to specific receptor in plasma membrane • Membrane invaginates, forming a vesicle surrounding the bound molecules (still on the outside) • Vesicle fuses with endosome and a lysozome Nucleic Acid Fundamentals

10. What happens next? • Inside the lysozome, the foreign material and the receptor get degraded • … or ligand or receptor or both get recycled Nucleic Acid Fundamentals

11. Example: LDL-cholesterol Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences Nucleic Acid Fundamentals

12. Exocytosis Diagram courtesy LinkPublishing.com • Materials to be secreted are enclosed in vesicles by the Golgi apparatus • Vesicles fuse with plasma membrane • Contents released into extracellular space Nucleic Acid Fundamentals

13. 6 1 5 Pyrimidines 4 2 3 • Single-ring nucleic acid bases • 6-atom ring; always two nitrogens in the ring, meta to one another • Based on pyrimidine, although pyrimidine itself is not a biologically important molecule • Biological pyrimidines have O’s and N’s attached • Tautomerization possible • Note plane of symmetry in pyrimidine structure Nucleic Acid Fundamentals

14. Uracil and thymine • Uracil is a simple dioxo derivative of pyrimidine: 2,4-dioxopyrimidine • Thymine is 5-methyluracil • Uracil is found in RNA;Thymine is found in DNA • Other tautomers: moving hydrogens among N’s and O’s Nucleic Acid Fundamentals

15. Tautomers • Lactam and Lactim forms • Getting these right was essential to Watson & Crick’s development of the DNA double helical model Nucleic Acid Fundamentals

16. Cytosine • This is 2-oxo,4-aminopyrimidine • The other pyrimidine base found in DNA & RNA • Spontaneous deamination (CU) involves replacing amine with hydroxyl • Again, other tautomers can be drawn Nucleic Acid Fundamentals

17. Cytosine:amino and imino forms • Again, this tautomerization needs to be kept in mind Nucleic Acid Fundamentals

18. 7 6 5 1 8 4 Purines 2 9 3 • Derivatives of purine; again, the root molecule isn’t biologically important • Six-membered ring looks a lot like pyrimidine • Numbering works somewhat differently: note that the glycosidic bonds will be to N9, whereas it’s to N1 in pyrimidines Nucleic Acid Fundamentals

19. Adenine • This is 6-aminopurine • Found in RNA and DNA • We’ve seen how important adenosine and its derivatives are in metabolism • Tautomerization happens here too Nucleic Acid Fundamentals

20. Guanine • This is 2-amino-6-oxopurine • Found in RNA, DNA • Lactam, lactim forms Nucleic Acid Fundamentals

21. Other natural purines • Hypoxanthine and xanthine are biosynthetic precursors of A & G • Urate is important in nitrogen excretion pathways Nucleic Acid Fundamentals

22. Tautomerization and H-bonds • Lactam forms predominate at neutral pH • This influences which bases are H-bond donors or acceptors • Amino groups in C, A, G make H-bonds • So do ring nitrogens at 3 in pyrimidines and 1 in purines • … and oxygens at 4 in U,T, 2 in C, 6 in G Nucleic Acid Fundamentals

23. iClicker quiz question 1 1. This is • (a) cytosine, amino form • (b) adenine, amino form • (c) cytosine, imino form • (d) adenine, imino form • (e) none of the above Nucleic Acid Fundamentals

24. iClicker quiz question 2 2. This is • (a) uracil, lactam form • (b) uracil, lactim form • (c) guanine, lactam form • (d) guanine, lactim form • (e) none of the above. Nucleic Acid Fundamentals

25. Nucleosides • As mentioned earlier, these are glycosides of the nucleic acid bases • Sugar is always ribose or deoxyribose • Connected nitrogen is: • N1 for pyrimidines (on 6-membered ring) • N9 for purines (on 5-membered ring) Nucleic Acid Fundamentals

26. Pyrimidine nucleosides • Drawn here in amino and lactam forms cytidine uridine Nucleic Acid Fundamentals

27. Pyrimidine deoxynucleosides 2’-deoxythymidine 2’-deoxycytidine 2’-deoxyuridine Nucleic Acid Fundamentals

28. A tricky / tedious nomenclature issue • Remember that thymidine and its phosphorylated derivatives ordinarily occur associated with deoxyribose, not ribose • Therefore many people leave off the deoxy- prefix in names of thymidine and its derivatives: it’s usually assumed. Nucleic Acid Fundamentals

29. Purine nucleosides • Drawn in amino and lactam forms adenosine guanosine Nucleic Acid Fundamentals

30. Purine deoxynucleosides 2’-deoxyguanosine 2’-deoxyadenosine Nucleic Acid Fundamentals

31. Glycosidic bonds • This illustrates the roughly perpendicular positionings of the base and sugar rings Nucleic Acid Fundamentals

32. Conformations around the glycosidic bond • Rotation of the base around the glycosidic bond is sterically hindered • In syn conformation: some interference between C-2 oxygen of the base and the sugar • Therefore pyrimidines are always anti, and purines are usually anti • Furanose and base rings are roughly perpendicular Nucleic Acid Fundamentals

33. Mono-phosphorylated nucleosides • We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: • They are nucleotides • Adenosine 5’-monophosphate = AMP = adenylate • GMP = guanylate; CMP = cytidylate; UMP = uridylate Nucleic Acid Fundamentals

34. Deoxynucleotides • Similar nomenclature • dAMP = deoxyadenylate • dGMP = deoxyguanylate • dCMP = deoxycytidylate • dTMP (= TMP) = deoxythymidylate = thymidylate Nucleic Acid Fundamentals

35. iClicker question 3 3. This is an example of a • (a) nucleobase • (b) nucleoside • (c) nucleotide • (d) deoxynucleotide • (e) none of the above Nucleic Acid Fundamentals

36. Di and triphosphates • Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety Nucleic Acid Fundamentals

37. Cyclic phospho-diesters • 3’ and 5’ hydroxyls are both involvedin -O-P-O bonds • cAMP and cGMP are the important ones(see earlier in the course!) Cyclic adenosine mono-phosphate Nucleic Acid Fundamentals

38. Chirality in nucleic acids • Bases themselves are achiral • 3/4 asymmetric centers in ribose • 2/3 in deoxyribose • Glycosidic bond gives us 1 more,so there are 4 for ribonucleosides,3 for deoxyribonucleosides • Same for nucleotides:phosphates don’t add asymmetries Nucleic Acid Fundamentals

39. Solubility of nucleosides and lability of glycosidic linkages • The sugar makes nucleosides more soluble than the free bases • Nucleosides are generally stable to basic hydrolysis at the glycosidic bond • Acid hydrolysis: • Purines: glycosidic bond fairly readily hydrolyzed • Pyrimidines: resistant to acid hydrolysis Nucleic Acid Fundamentals

40. pKa’s for base N’s and PO4’s Nucleic Acid Fundamentals

41. UV absorbance • These aromatic rings absorb around 260 Nucleic Acid Fundamentals

42. Hydrolysis of nucleic acids • RNA & DNA are susceptible to [enzymatic] degradation at the phosphodiester linkages and on the glycosidic linkage • RNA can be cleaved spontaneously through 3’-2’ cyclization • Then we’ll go on to the enzymatic hydrolyses Nucleic Acid Fundamentals

43. Why alkaline hydrolysis of RNA works • Cyclic phosphate intermediate stabilizes cleavage product Nucleic Acid Fundamentals

44. The cyclic intermediate • Hydroxyl or water can attack five-membered P-containing ring on either side and leave the –OP on 2’ or on 3’. • If it stays on 2’, we can’t keep the chain going! Nucleic Acid Fundamentals

45. Enzymatic cleavage of oligo- and polynucleotides • Enzymes are phosphodiesterases • Could happen on either side of the P • 3’ cleavage is a-site; 5’ is b-site. • Endonucleases cleave somewhere on the interior of an oligo- or polynucleotide • Exonucleases cleave off the terminal nucleotide Nucleic Acid Fundamentals

46. Specificity in nucleases • Some cleave only RNA, others only DNA, some both • Often a preference for a specific base or even a particular 4-8 nucleotide sequence (restriction endonucleases) • These can be used as lab tools, but they evolved for internal reasons Nucleic Acid Fundamentals

47. Restriction endonucleases • Evolve in bacteria as antiviral tools • “Restriction” because they restrict the incorporation of foreign DNA into the bacterial chromosome • Recognize and bind to specific palindromic DNA sequences and cleave them Nucleic Acid Fundamentals

48. Restriction Endonucleases II • Self-cleavage avoided by methylation • Types I, II, III: II is most important • I and III have inherent methylase activity; II has methylase activity in an attendant enzyme Nucleic Acid Fundamentals

49. What do we mean by palindromic? • In ordinary language, it means a phrase that reads the same forward and back: • Madam, I’m Adam. (Genesis 3:20) • Eve, man, am Eve. • Sex at noon taxes. • Able was I ere I saw Elba. (Napoleon) • A man, a plan, a canal: Panama! (T. Roosevelt) • With DNA it means the double-stranded sequence is identical on both strands Nucleic Acid Fundamentals

50. Palindromic DNA • G-A-A-T-T-C • Single strand isn’t symmetric: but the combination with the complementary strand is: • G-A-A-T-T-CC-T-T-A-A-G • These kinds of sequences are the recognition sites for restriction endonucleases. This particular hexanucleotide is the recognition sequence for EcoRI. Nucleic Acid Fundamentals