CHM 3240 – Biochemistry

1. CHM 3240 – Biochemistry Instructor - Paul Stein S3305 phone = 6065 email = pstein@css.edu Meeting Times- MWF 9:15 - 10:20 - S2316 Office Hours: MWF 12:30 – 1:45, T 11:00 – 12:00, R 1:00 – 2:00 Please feel free to make an appointment or drop in at any time. Title: Lehninger Principles of Biochemistry Author: Nelson and CoxISBN: 1429234148 Required http://faculty.css.edu/pstein/steinhome.htm Today’s topics: amino acids – general structure variations among 20 amino acids classification by polarity/ionization acid/base properties

2. Protein = Polymers of Amino Acids Amino Acid H | H2N - Ca - COOH | R amino group Carboxyl group a-carbon Side chain If R = -CH3 (ALA), is Ca chiral? a) yes b) no

3. All amino acids are chiral (except glycine) Proteins only contain L amino acids These are all S except for Cys which is R

4. Sidechain Categories Nonpolar: hydrocarbon + Met (C-S-C) Polar: hydroxyl (OH) Cys (S-H) O amide || - C – NH2 Acidic: O carboxylic Acid || - C - OH Basic: Amine (-NH2)

5. Nonpolar R Groups

6. These amino acids side chains can form hydrogen bonds. Cysteine can form disulfide bonds.

7. Acidic Amino Acids Shown in ionized form – dominant at pH 7.4

8. Basic Amino Acids Shown in dominant form at pH 7.4

9. nonpolar polar Polar with some NP character These amino acid side chains absorb UV light at 270–280 nm

10. What is Beer’s Law? Proteins are typically detected by their absorbance (A280). Beer’s Law: A = ebC applies. e is determined mostly by the # or Trp (& Tyr) residues in the protein.

11. Review Questions – amino acids Which functional group is not part of polar side chains. A. -COOH O || B. – C – NH2 C. -OH All natural amino acids in proteins are? A. nonpolar B. L stereoisomers C. S stereoisomers D. g-amino acids The Val side chain …. -CH2 – CH3 is ……? | CH3 A. Nonpolar B. Polar C. Acidic D. Basic

12. Essential − Required in your dietary protein Can’t be synthesized from other dietary sources (in bolded print on amino acid handout) Nonessential − Not required in diet Can be made from carbohydrates + N from other amino acids. The RDI (recommended daily intake) of protein (~ 60 g), requires that all essential amino acids are represented in significant amounts.

13. Amino Acid – acid/base properties Write an equation showing –COOH acting as an acid. Write the equation representing Ka for this reaction. -NH2+ H+ ↔ -NH3+ base acid Ka = [-NH2] [H+] [-NH3+] -COOH ↔ -COO- + H+ acid base Ka = [-COO-] [H+] [-COOH] H | H2N - Ca - COOH | CH3 pKa = - log Ka

14. Amino Acid (Ala) – acid/base properties pH = pK + log (b/a) Both groups > 90% ionized between ~ pH 3 – 8.5 pK= 9.7 see column pK amino on aa handout H | H2N - Ca - COOH | CH3 pK= 2.3 see column pKcarboxy on aa handout + H3N ― Acidic and basic side chains may also be ionized with the fraction ionization determined by the HH equation. (see column pK side chain on aa handout)

15. Low pH Neutral pH High pH pK ~ 2 pK ~ 9 pH = pK + log (b/a) Non-ionizable side chain

16. Side Chain ionization Aspartic acid – pK = 4.1 What fraction is in acid vs. base form at pH = 5.0 O || — C — OH ↔ acid (a) base (b) O || — C — O- + H+ 11% acid 89% base pH = pK + log (b/a) 5.0 = 4.1 + log (b/a) log (b/a) = 5.0 – 4.1 = 0.9 b/a = 10log (b/a) = 100.9 = 7.9 or 7.9:1 Fraction base = 7.9/(1+7.9) = 0.89 or 89% base

17. Side Chain ionization Aspartic acid – pK = 4.1 What fraction is in acid form at pH = 3.6 a) 0.50 b) 0.24 c) 0.32 d) 0.76 O || — C — OH ↔ acid (a) base (b) O || — C — O- + H+ 76% acid 24% base pH = pK + log (b/a) 3.6 = 4.1 + log (b/a) log (b/a) = 3.6 – 4.1 = -0.5 b/a = 10log (b/a) = 10-0.5 = 0.32 or 0.32:1 Fraction base = 0.32/(1.32) = 0.24 or 24% base

18. Qualitative Estimate Henderson HasselbachEquation Each pH unit variation from pK shifts equilibrium 10x

19. Peptide Bond O H H || -N - C - C - OH | R2 O H H || H-N - C - C | R1 - OH H O H H || H-N - C - C | R1 O H H || -N - C - C - OH | R2 Dipeptide + HOH

20. AMINO END OOOO H ||HH ||HH ||HH ||_ H3N-C-C-N-C-C-N-C-C-N-C-C-O | ||| R1R2R3R4 + CARBOXY END POLYPEPTIDE CHAIN Sequence (primary structure) = R1-R2-R3-R4 …. Etc.

21. Sequence = YGGFL Listed order of aas from amino end to carboxyl end Note bond angles make extended backbone ‘zig-zag’ and side chains alternate sides in extended chain

22. Peptide ― A polymer composed of a small number of amino acids. dipeptide, tripeptide, oligopeptide, polypeptide Peptides lack (too small) a specific folded structure. Peptide examples Glutathione (ROS protection) = gGlu-Cys-Gly LeuEnkephalin (neurotransmitter) =YGGFL Aspartame (artificial sweetener) = Asp-Phe-O-CH3

23. Protein = Polymers of Amino Acids Protein ― A molecule composed of 1 or more folded polypeptide chains that performs a biochemical function. Myoglobin sequence 153 aa; 17053 MW; GLSDGEWQLV LNVWGKVEAD IPGHGQEVLI RLFKGHPETL EKFDKFKHLK SEDEMKASED LKKHGATVLT ALGGILKKKG HHEAEIKPLA QSHATKHKIP VKYLEFISEC IIQVLQSKHP GDFGADAQGA MNKALELFRK DMASNYKELG FQG e280 = 13,980 M-1 cm-1. pI = 7.3

24. Key questions about Proteins What is its sequence and amino acid composition? What is its three-dimensional structure? (x-ray crystallography or 2D NMR) What is its function? What is its mechanism of action? How is its function regulated? How is it related to other proteins? Where is it localized within the cell? What are its properties? MW, pI, e280, etc.

25. Studying Proteins requires separation • Separation relies on differences in physical and chemical properties • Charge (depends on pH: pI = pH at which it has 0 charge) • Size (MW 1 Dalton = 1000 g/mol) • Affinity for a ligand (related to function) • Solubility • Hydrophobicity • Thermal stability • Chromatography is commonly used for preparative separation

26. CHROMATOGRAPHY Mobile Phase — solvent + solutes to be separated. Stationary Phase — solid support matrix solutes have differential adherence to matrix. Chromatography Types Adsorption (nonspecific VdW) Gel Filtration (size exclusion) Ion Exchange (charge) Affinity (specific ligand)

27. Protein = Polymers of Amino Acids Protein ― A molecule composed of 1 or more folded polypeptide chains that performs a biochemical function. Myoglobin sequence 153 aa; 17053 MW; GLSDGEWQLV LNVWGKVEAD IPGHGQEVLI RLFKGHPETL EKFDKFKHLK SEDEMKASED LKKHGATVLT ALGGILKKKG HHEAEIKPLA QSHATKHKIP VKYLEFISEC IIQVLQSKHP GDFGADAQGA MNKALELFRK DMASNYKELG FQG pI = 7.3 e280 = 13,980 M-1 cm-1. Separation relies on differences in physical and chemical properties.

28. Column Chromatography

29. Separation by Size Large MW standard (blue dextran) = void volume A280 Fraction # Larger proteins Smaller proteins

30. Separation by Charge If pH > pI then the protein has a negative charge (-). If pH = pI then the protein has No net charge, such that the # of (+) groups = # (-) groups. If pH < pI then the protein has a positive charge (+).

31. Polymerized (and cross-linked) acrylamide is used for protein electrophoresis (and DNA sequencing)

32. PAGE = Polyacrylamide Gel Electrophoresis Apply protein mixture to wells. Apply voltage to system. (-) proteins migrate toward (+) electrode. Different proteins migrate at different rates dependent on charge:size ratio. v = Ez/f

33. Types of PAGE Type SeparationDetermines standard charge “pattern” size SDS size MW Isoelectric charge pI focusing v = Ez/f V = migration rate E = voltage applied (same for all proteins in mixture) Z = protein charge (related to pH and pI) F = frictional coefficient (related to protein size and gel density (constant).

34. SDS PAGE: Molecular Weight • SDS – sodium dodecyl sulfate – a detergent • SDS micelles bind to and unfold all the proteins • SDS gives all proteins an uniformly negative charge with similar shape. • Rate of movement will only depend on size: small proteins will move faster.

35. SDS PAGE: Separates proteins based on size. v = Ez/f all proteins are uniformly (-) but larger proteins have large “f” frictional component which slows down their migration.

36. MW ~ 40,000