1. The three important structural features of proteins: a. Primary (1 o ) – The amino acid sequence (coded by genes) b. Secondary (2 o ) – The interaction of amino acids that are close together or far apart in the sequence c. Tertiary (3 o ) – The interaction of amino acids that are far apart in sequence In 2 o and 3 o the primary interaction is noncovalent Some proteins have quaternary structure (4 o ): noncovalent interaction of multiple polypeptide chains (subunits) Native structure (conformation)  biological function

3. Peptide bonds link amino acids in proteins Figure 4.1 Amino-terminus Carboxyl- terminus Residue or side chain

4. Alanine Ala (A) Serine Ser (S) Dipeptide Ala-Ser or AS Peptide bonds link amino acids in proteins Primary sequence

5. Peptide bonds link amino acids in proteins Primary sequence has directionality Important: the sequence Tyr-Gly-Gly-Phe-Leu is not the same as Leu-Phe-Gly-Gly-Tyr Figure 4.2

6. Figure 4.3-the polypeptide backbone is rich With hydrogen bond donors and acceptors How many amino acids are typically found in polypeptide chains? 1 amino acid molecular weight is ~110 g/mol or 110 Da (Daltons)

7. Proteins can be very large, hundreds of amino acids long The enzyme HMG-CoA reductase MLSRLFRMHGLFVASHPWEVIVGTVTLTICMMSMNMFTGNNKICGWNYECPK FEEDVLSSDIIILTITRCIAILYIYFQFQNLRQLGSKYILGIAGLFTIFSSFVFSTVVIH FLDKELTGLNEALPFFLLLIDLSRASTLAKFALSSNSQDEVRENIARGMAILGPTF TLDALVECLVIGVGTMSGVRQLEIMCCFGCMSVLANYFVFMTFFPACVSLVLEL SRESREGRPIWQLSHFARVLEEEENKPNPVTQRVKMIMSLGLVLVHAHSRWIAD PSPQNSTADTSKVSLGLDENVSKRIEPSVSLWQFYLSKMISMDIEQVITLSLALL LAVKYIFFEQTETESTLSLKNPITSPVVTQKKVPDNCCRREPMLVRNNQKCDSV EEETGINRERKVEVIKPLVAETDTPNRATFVVGNSSLLDTSSVLVTQEPEIELPRE PRPNEECLQILGNAEKGAKFLSDAEIIQLVNAKHIPAYKLETLMETHERGVSIRR QLLSKKLSEPSSLQYLPYRDYNYSLVMGACCENVIGYMPIPVGVAGPLCLDEKE FQVPMATTEGCLVASTNRGCRAIGLGGGASSRVLADGMTRGPVVRLPRACDSA EVKAWLETSEGFAVIKEAFDSTSRFARLQKLHTSIAGRNLYIRFQSRSGDAMGM NMISKGTEKALSKLHEYFPEMQILAVSGNYCTDKKPAAINWIEGRGKSVVCEA VIPAKVVREVLKTTTEAMIEVNINKNLVGSAMAGSIGGYNAHAANIVTAIYIAC GQDAAQNVGSSNCITLMEASGPTNEDLYISCTMPSIEIGTVGGGTNLLPQQACL QMLGVQGACKDNPGENARQLARIVCGTVMAGELSLMAALAAGHLVKSHMIH NRSKINLQDLQGACTKKTA

8. Practice Problem Draw the chemical structure of the tripeptide Glu – Ser – Cys at pH 7. Answer the following with regard to this tripeptide: 1. Indicate the charge present on any ionizable group(s). 2. Indicate, using an arrow, which covalent bond is the peptide bond. 3. What is the net, overall charge of this tripeptide at pH 7? __________ 4. What is this peptide called using the one-letter code system for amino acids? ______

9. Double bond character of the peptide bond Bond lengths reveal C-N is between a single and a double bond. (Figure 4.7)

10. Trans and Cis conformations of a peptide group Figure 4.8 Nearly all peptide groups in proteins are in the trans conformation

11. The N-C  and C  -CO bonds are not rigid and rotation is possible Figure 4.9 Phi angle Psi angle CC Are all angles “allowed”?

12. Ramachandran Plot Figure 4.10

13. The amino acid cysteine also stabilizes proteins through the formation of a disulfide bond. Figure 4.4 Insulin Figure 4.5

14. Secondary structure of proteins Alpha helix Pitch is ~5.4 Å or 3.6 AAs

15. The coil in the alpha helix allows for Hydrogen bonding Figure 4.12

16. The stability of the alpha helix is dependent upon the residues attached. Gly and Pro are not prevalent in most  -helix

17. The alpha helix can sometimes be amphipathic.

18. Amphipathic  -helices are often Found on the surface of proteins hydrophilic hydrophobic A dehydrogenase globular protein

19. Secondary Structure – the Beta (  ) sheet or Beta strand Figure 4.15- the peptide chain is more elongated than In the alpha helix.

20. Secondary Structure – the Beta (  ) sheet or Beta strand Antiparallel N C

21. Secondary Structure – the Beta (  ) sheet or Beta strand Parallel C C

22. Figure 4.17- both types of  -sheets are possible in one protein. C C N

23. Figure 4.18  -sheets can be found with a twist

24. The beta sheet. Side chains alternate from one side to another

25. The ability for polypeptides to reverse direction requires reverse turns and/or loops Figure 4.19 A protein involved in Fatty acid metabolism

26. Reverse Turns and loops Figure 4.20 Type I  turn Hydrogen bonding

27. Tertiary Structure of Proteins Supersecondary structures often called “motifs” Figure 4.27

28. Tertiary Structure of Proteins Domains are a combination of motifs Figure 4.28 Protein found on surface of some Immune system cells

29. Tertiary structure of proteins Domains in Pyruvate kinase this protein has 3 domains

30.  -Keratin: A fibrous protein with extensive secondary structure Figure 4.21-A coiled coil protein

31. Collagen -25% to 35% total protein in mammals -Fibrous protein found in vertebrate connective tissue (skin, bone, teeth) - Triple helix structure Strength is greater than steel of equal cross section -only 3 amino acids per turn Figure 4.24. A super helical structure

32. Collagen is 35% Glycine 21% Proline + Hydroxyproline The repeating unit is Gly – X – Y X is usually Pro Y is usually Hyp triple helix is packed with Glycines (red)

33. 4-hydroxyproline For every Gly-X-Y, there is one interchain Hydrogen bond (between chains). Read Clinical Insight (pg 55)– Osteogenesis Imperfecta and Scurvy

34. Figure 4.25 - Myoglobin (153 amino acids) Globular Proteins- very compact and water soluble WHY?

35. Figure 4.26 - Distribution of amino acids in myoglobin Charged amino acids (blue) Hydrophobic amino acids (yellow) Surface Interior

36. Quaternary Structure-multiple polypeptide strands Intermingle though noncovalent interactions. Figure 4.29 A dimer of two subunits (polypeptides)

37. Figure 4.30 Hemoglobin: a tetramer protein This protein has primary, secondary tertiary and quaternary structures

38. How do proteins fold and unfold? The information for proteins to fold is contained in the amino acid sequence. Can proteins fold by themselves or do they need help? Is there a way in which we can predict from the primary sequence how a protein will fold??

39. First, we must denature a protein and see if it will spontaneously refold to the native structure How can we denature proteins? a. Reducing agents 2-mercaptoethanol  break disulfide bonds b. heat c. acids or bases d. heavy metals (good Lewis acids  bind to cysteine) e. chaotropic agent-Urea (help weaken hydrogen bonding and eventually disrupt hydrophobic core. )

40. Figure 4.31 – 4 cystine residues in bovine ribonuclease A

41. Anfinsen’s protein folding Experiment. Figure 4-32 Denature Protein with  -mercaptoethanol and Urea.

42. Anfinsen result after removal of urea and most of the  -mercaptoethanol Enzyme slowly regains activity!! Native conformation is re-established Conclusion: primary sequence specifies conformation

43. Figure 4.35 Energy well of cooperative folding Protein folding is very fast! ~ large Proteins may take ~ hrs, but smaller Proteins may fold in one step. Read Clinical Insight Amyloid fibrils and prion diseases (pg 61)

44. Assignment Read Chapter 4 Read Chapter 6 Topics not covered: Chapter 5