Biomolecules: Peptides and Proteins

1. Biomolecules:Peptides and Proteins Lecture 5, Medical Biochemistry

2. Lecture 5 Outline • Overview of amino acids, peptides and the peptide bond • Discuss the levels of protein structure • Describe techniques used for analysis of proteins

3. Planar nature of the peptide bond. The partial double bond characteristic prevents free rotation around the C-N bond; keeping it in the same plane with the attached O and H atoms. These planar bonds can pivot around the shared Ca atom

4. Levels of Protein Structure

5. Protein Structure Levels • PRIMARY: the linear sequence of amino acids linked together by peptide bonds • SECONDARY: regions within polypeptide chains with regular, recurring, localized structure stabilized by H-bonding between constituent amino acid residues

6. Protein Structure Levels (cont) • TERTIARY: the overall three-dimensional conformation of a protein • QUATERNARY: the three-dimensional conformation of a protein composed of multiple polypeptide subunits • THE PRIMARY AMINO ACID SEQUENCE IS THE ULTIMATE DETERMINANT OF FINAL PROTEIN STRUCTURE

7. Disulfide bonds Form between two intra- or interchain cysteine residues, product called cystine - Stabilizes/creates protein conformation - Prevalent in extracellular/ secreted proteins Ex: INSULIN

8. Stabilizing Forces 1. Electrostatic/ionic 3. Hydrophobic interactions 2. Hydrogen bonds 4. Disulfide bonds

10. 2o Structure: a-helix each oxygen of a carbonyl group of a peptide bond forms a H-bond with the hydrogen atom attached to a nitrogen in a peptide bond 4 amino acids further along the chain; very stable structurally; prolines will disrupt helix formation

11. End-on view of a-helix

12. b-sheet In this secondary structure, each amino acid residue is rotated 180o relative to its adjacent residue. Occur most commonly in anti-parallel directions, but can also be found in parallel. H-bonds between adjacent chains aid in stabilizing the conformation. Anti-Parallel Parallel

13. Super-secondary structure examples b-bend

14. Super-secondary structures commonly found in some DNA-binding proteins

15. Domains, examples: b-Barrel Bundle Saddle

16. Ex: Tertiary Structure Ex: Quaternary Structure

17. Myoglobin b-subunit Hemoglobin

18. Structure of Myoglobin and Hemoglobin • The amino acid sequences of myoglobin and hemoglobin are similar (or, highly conserved) but not identical • Their polypeptide chains fold in a similar manner • Myoglobin is found in muscles as a monomeric protein; hemoglobins are found in mature erythrocytes as multi-subunit tetrameric proteins. Both are localized to the cytosol

19. Sequence Comparison Examples (Surface helix) Myoglobin Hba (horse) Hbb (horse) Hba (human) Hbb (human) Hbg (human) Hbd (human) (Internal helix) Myoglobin Hba (horse) Hbb (horse) Hba (human) Hbb (human) Hbg (human) Hbd (human)

20. Myoglobin Properties • At the tertiary level, surface residues prevent one myoglobin from binding complementarily with another myoglobin; thus it only exists as a monomer. • Each monomer contains a heme prosthetic group: a protoporphryin IX derivative with a bound Fe2+ atom. • Can only bind one oxygen (O2) per monomer • The normal physiological [O2] at the muscle is high enough to saturate O2 binding of myoglobin.

21. Heme Structure Protein-Heme Complex with bound oxygen Heme-Fe2+

22. Hemoglobin Properties • At the tertiary level, the surface residues of the a and b subunits form complementary sites that promote tetramer formation (a2b2), the normal physiological form of hemoglobin. • Contains 4 heme groups, so up to 4 O2 can be bound • Its physiological role is as a carrier/transporter of oxygen from the lungs to the rest of the body, therefore its oxygen binding affinity is much lower than that of myoglobin. • If the Fe2+ becomes oxidized to Fe3+ by chemicals or oxidants, oxygen can no longer bind, called Methemoglobin

23. Biochemical Methods to Analyze Proteins • Electrophoresis • Chromatography: Gel filtration, ion exchange, affinity • Mass Spectrometry, X-ray Crystallography, NMR • You will not be tested on the sections in your textbook describing amino acid separations (Ch 4), peptide/protein sequencing and synthesis (Ch 5), and X-ray crystallography/NMR (Ch 6)

24. Protein Separation by SDS-Polyacrylamide Gel Electrophoresis Presence of SDS, a detergent, denatures and linearizes a protein (Na and sulfate bind to charged amino acids, the hydrocarbon chain interacts with hydrophobic residues). An applied electric field leads to separation of proteins based on size through a defined gel pore matrix. For electrophoresis in the absence of SDS, separation is based on size, charge and shape of the protein (proteins are not denatured and can potentially retain function or activity)

25. SDS-Polyacrylamide Gel (cont) Separation of proteins based on their size is linear in relation to the distance migrated in the gel. Using protein standards of known mass and staining of the separated proteins with dye, the mass of the proteins in the sample can be determined. This is useful for purification and diagnostic purposes.

26. Gel filtration Separation is based on protein size. Dextran or polyacrylamide beads of uniform diameter are manufactured with different pore sizes. Depending on the sizes of the proteins to be separated, they will enter the pore if small enough, or be excluded if they are too large. Hydrophobic Chromatography Proteins are separated based on their net content of hydrophobic amino acids. A hydrocarbon chain of 4-16 carbons is the usual type of resin.

27. Ion Exchange Chromatography Separation of proteins based on the net charge of their constituent amino acids. Different salt concentrations can be used to elute the bound proteins into tubes in a fraction collector. As shown below, resins for binding (+) or (-) charged proteins can be used

28. Affinity Chromatography • Based on the target proteins ability to bind a specific ligand, only proteins that bind to this ligand will be retained on the column bead. This is especially useful for immunoaffinity purification of proteins using specific antibodies for them. • Example:

29. Protein Structure Methods • The sequence of a protein (or peptide) is determined using sophisticated Mass Spectrometry procedures. The three dimensional structures of proteins are determined using X-ray crystallographic and NMR (nuclear magnetic resonance) spectroscopic methods. • Protein sequence data banks useful for structural and sequence comparisons • Please note that the new discipline termed “Proteomics” is evolving to incorporate cross-over analysis of sequence data banks, Mass Spec methodology, and living cells