TOPICS IN (NANO) BIOTECHNOLOGY Lecture 4

1. PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Lecture 4 27th April, 2005

2. Let’s recap...

3. microvilli Golgi Body Rough Endoplasmic Reticulum Mitochondria Vacuole Cytosol Smooth Endoplasmic Reticulum Ribosome Nucleolus Chromatin Nucleus Cell (plasma) Membrane The Cell

4. DNA packaging

5. The Central Dogma

6. RNA Processing/Splicing

7. Transcription

8. Translation

9. Translation

10. The Genetic Code

13. Protein synthesis

14. Proteins - proteins are the most diverse molecule (polymer) in existence - this diversity underlies their function

15. Diversity - proteins are the most diverse molecule (polymer) in existence and this diversity underlies their function - diversity comes from the 20 different monomeric units (amino-acids) which comprise proteins - the average protein in a bacterium is 150 ± 87 amino acids.

16. Diversity Consider the number of combinations for a 100 amino acid protein = 20100 <--- that’s large number ! Definitions: amino-acid the monomer or basic building block peptide 2-20 amino acids (a small protein) polypeptide/protein 20-2000 amino acids

17. Amino acids H O H C C N H OH H acid group (pK2 ~ 3 ) amino group (pK1 ~ 9 ) Glycine (the simplest amino acid) pI (isoelectric point) = pK1 + pK2 = (3 + 9)/2 = 6 at pH 6, glycine is neutral

18. Amino acids H +H3N C COO– H acid group (pK2 ~ 3 ) amino group (pK1 ~ 9 ) Glycine (the simplest amino acid) pI (isoelectric point) = pK1 + pK2 = (3 + 9)/2 = 6 at pH 6, glycine is neutral

19. Amino acids H +H3N C COO– R glycine (the simplest amino acid) R = H

20. Amino acids H +H3N C COO– R Diversity in the chemical behavior of amino acids comes from the chemical nature of the R group R = nonpolar (hydrophobic — carbon chains) R = polar (hydrophilic — substituents that can hydrogen bond) R = charged group (acidic or basic) The R group is commonly known as an amino acid sidechain

21. Amino acids H +H3N C COO– CH3 alanine (a polar amino acid) R = CH3 (a methyl functional group)

22. Naming amino acids • proton/hydrogen a carbon carbonyl oxygen H amino hydrogen or amino proton +H3N C COO– CH3 carbonyl carbon amino nitrogen  carbon alanine (a polar amino acid) R = CH3 (a methyl functional group)

23. Non-polar amino acids - R group consists of carbon chains leucine and isoleucine are structural isomers

24. Non-polar amino acids - R group consists of carbon chains phenylalanine and tryptophan have aromatic rings which are flat due to the double bond network proline has its R group bound to the amino nitrogen to form a ring network Methionine has a sulphur atom in its sidechain sulphur has the same valence as oxygen

25. Polar amino acids - R group consists of carbon, oxygen and nitrogen atoms - together they make the sidechain more hydrophilic Ser and thr are a mix of carbon chains and hydroxyl functional groups (-OH). Cysteine has a thiol group (-SH) which is otherwise structurally similar to serine but not chemically similar Asn and gln have an amide functional group

26. Polar amino acids - R group has a charge at physiological pH (7.4). pK of the charged groups vary carboxyl group carboxyl group guanidinio group imidazole group amino group

27. Describing amino acids - amino acids have a full name (glycine), a short three-letter name (gly) and an even shorter one-letter name (G) A ala alanine C cys cysteine D asp aspartic acid E glu glutamic acid F phe phenylalanine G gly glycine H his histidine I ile isoleucine K lys lysine L leu leucine M met methionine N asn asparagine P pro proline Q gln glutamine R arg arginine S ser serine T thr threonine V val valine W trp tryptophan Y tyr tyrosine nonpolar polar acidic (negative charge) basic (positive charge)

28. L- vs D- configuration

29. The Peptide Bond

30. Joining amino acids - in a cell, a complex assembly of proteins and RNA called a ribosome catalyse a dehydration reaction (loss of water) to join amino acids together chain extends in this direction The ribosome only joins new amino acids to this end (the carboxy end) The ribosome does not join an amino acid to this end (the amino end) loss of water

31. Joining amino acids - a peptide bond (like an amide bond C-O-N) joins each amino acid - the invariant purple part of the polypeptide is generally called the backbone - it’s the sidechains that give a protein its unique chemical character

32. Protein architecture Primary structure Secondary structure Tertiary structure Quaternary structure

33. Protein architecture

34. Primary Structure - lysozyme is a protein found in egg white that has anti-bacterial properties. It is an enzyme which catalyses the breakdown of a polysaccharide network necessary to maintain the integrity of the bacterium. - there are 129 amino acids (or residues) in lysozyme. The amino and carboxyl ends are free (not bound to anything else) - the sequence of amino acids is called the primary structure

35. N–H ------ O=C d+  hydrogen bond 1.8 to 2.4 Å in length Secondary Structure Much farther than 2.4 Å so the protein folds up to make H-bonds - the protein spontaneously folds to minimize hydrophobic (nonpolar) sidechain exposure to water and maximize hydrophilic (polar and charged) sidechain exposure to water. - the HN (amide) and CO (carbonyl) groups of the backbones have covalent bonds which are polarized much like water - the protein also folds up to encourage a hydrogen bond between the the HN and CO groups

36. residue n residue n+4 residue n+8 Secondary Structure - the alpha helix (a-helix) is one common form of secondary structure - much like the coils of a telephone cable - protein helices are always right-handed (look down the helix in this figure) - due to the hydrogen bonding network in an alpha helix, this structure is stable

38. Secondary Structure - the beta sheet (-sheet) is another common form of secondary structure much like the pleats of an accordion - beta sheets can join very distant parts of the protein together - due to the hydrogen bonding network, beta sheets are very stable

39. extended loop -sheet a-helix Tertiary Structure - the protein spontaneously folds to minimize hydrophobic (nonpolar) sidechain exposure to water and maximize hydrophilic (polar and charged) sidechain exposure to water.

40. Quaternary Structure - the active configuration of protein may consist of more than one folded protein unit - three collagen chains twist into a strong fiber

41. Quaternary Structure - the active configuration of protein may consist of more than one folded protein unit - three collagen chains twist into a strong fiber - two alpha subunits and two beta subunits combine to form a functional molecule of hemoglobin. Each subunit bind one molecule of heme, an iron containing cofactor which helps bind oxygen

42. Protein folding - in addition to hydrogen bonds and the force to minimize the exposure of hydrophobic amino acid sidechains, there are other mechanisms that assist folding - disulfide bonds occur between two cysteines - a positively charged sidechain may form an ionic bond with a negatively charge sidechain (lysine -> aspartate)

43. Protein folding • - temperature (heat), pH and solvent conditions can be adjusted to unfold a protein back into a more extended form. • - when the unfolding conditions are reverted, many proteins have enough information stored in their sequence of amino acids to refold back to exactly the same tertiary structure. Other proteins get stuck along the way (curdled milk stays curdled after heat/cool treatment) • much research is done to solve the protein folding problem, or given a sequence, can one predict how the protein will fold up. • http://www.sumanasinc.com/webcontent/anisamples/nonmajorsbiology/proteinstructure.html

44. Mutatation • Mutations change the sequence of DNA • Mutations can be spontaneous or induced

45. Sickle Cell Anaemia - sickle cell anemia is caused by a point mutation in hemoglobin b chain (a is unaffected) val-his-leu-thr-pro-glu-glu … normal individual val-his-leu-thr-pro-val-glu … affected individual - only one amino acid is changed in the entire sequence of the protein glutamic acid sidechain -CH2-CH2-COO– acidic sidechain valine sidechain -CH-(CH3)2 nonpolar sidechain - the hemoglobin molecule folds up and functions (binds oxygen) but the mutation caused the protein to clump up in the cells. The clumping up distorts the cell shape and makes them architecturally weaker.

46. b  Sickle Cell Anaemia - the surface of the protein has sidechains sticking out. Polar and charged sidechains help the protein stay dissolved in water - the glutamic acid to valine mutation is a surface mutation

47. Mutations - mutations are responsible for numerous diseases - cystic fibrosis (point mutation) - Huntington’s disease (insertion of extra amino acids) - HIV uses mutations to its advantage - a drug that binds to an HIV protein may not bind very well only a few viral generations later - structural biologists study the relationship between protein structure and protein function - to design new or better drugs - to understand how proteins are constructed - (nature tends to use the same motif over and over again)

48. Modular nature of proteins - a single polypeptide chain often consists of a number of smaller autonomously folding units called domains. Sometimes they arranged like beads on a string… activity 3 H3N activity 1 COOH activity 2 - often though, each domain interacts with the others - much like quaternary structure built into ternary structure - over evolutionary time, the genes that encode each module/domain get shuffled and spliced to make new proteins activity 1 activity 3 activity 2

49. An important set of proteins: Enzymes

50. Enzymes • Thousands of biochemical reactions proceed at any given instant within living cells. These reactions are catalyzed by enzymes; • Enzymes are mostly proteins. But two important enzymes are most certainly to be RNA (ribozymes). One is the ribosome (peptidyl transfer) and the other is the spliceosome (splicing of intron); • Enzymes are the agents of metabolic function. Enzymes play key functions in controlling rate of reaction, coupling reactions, and sensing the momentary metabolic needs of the cell.