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amino asid,peptides and proteins
- 1. CHAPTER 3 Amino Acids, Peptides, Proteins • Structure and naming of amino acids • Structure and properties of peptides • Ionization behavior of amino acids and peptides • Purification and assay methods • Peptide sequencing and chemical synthesis • Protein sequence analysis
- 2. Proteins: Main Agents of Biological Function • Catalysis: –enolase (in the glycolytic pathway) –DNA polymerase (in DNA replication) • Transport: –hemoglobin (transports O2 in the blood) –lactose permease (transports lactose across the cell membrane) • Structure: –collagen (connective tissue) –keratin (hair, nails, feathers, horns) • Motion: –myosin (muscle tissue) –actin (muscle tissue, cell motility)
- 3. Amino Acids: Building Blocks of Protein • Proteins are heteropolymers of α-amino acids • Amino acids have properties that are well suited to carry out a variety of biological functions: – Capacity to polymerize – Useful acid-base properties – Varied physical properties – Varied chemical functionality
- 4. Amino Acids: Atom Naming • Organic nomenclature: start from one end • Biochemical designation: start from α-carbon and go down the R-group
- 5. Most α-Amino Acids are Chiral • The α-carbon has always four substituents and is tetrahedral • All (except proline) have an acidic carboxyl group, a basic amino group, and an alpha hydrogen connected to the α-carbon • Each amino acid has an unique fourth substituent R • In glycine, the fourth substituent is also hydrogen
- 6. Amino Acids: Classification Common amino acids can be placed in five basic groups depending on their R substituents: • Nonpolar, aliphatic (7) • Aromatic (3) • Polar, uncharged (5) • Positively charged (3) • Negatively charged (2)
- 7. Aliphatic Amino Acids • http://en.wikipedia.org/wiki/File:Aa.svg
- 8. Aromatic Amino Acids • http://en.wikipedia.org/wiki/File:Aa.svg
- 9. Charged Amino Acids • http://en.wikipedia.org/wiki/File:Aa.svg
- 10. Polar Amino Acids • http://en.wikipedia.org/wiki/File:Aa.svg
- 11. Special Amino Acids • http://en.wikipedia.org/wiki/File:Aa.svg
- 13. Not incorporated by ribosomes Arise by post-translational modifications of proteins Reversible modifications, esp. phosphorylation is important in regulation and signaling Uncommon Amino Acids in Proteins
- 14. The Genetic Code is organized by Amino Acid Properties
- 15. Ionization At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions At alkaline pH, the amino group is neutral –NH2 and the amino acid is in the anionic form.
- 16. Substituent effects on pKa Values α-carboxy group is much more acidic than in carboxylic acids α-amino group is slightly less basic than in amines
- 17. Amino Acids Can Act as Buffers Amino acids with uncharged side-chains, such as glycine, have two pKa values: The pKa of the α-carboxyl group is 2.34 The pKa of the α-amino group is 9.6 It can act as a buffer in two pH regimes.
- 18. Amino Acids Carry a Net Charge of Zero at a Specific pH •Zwitterions predominate at pH values between the pKa values of amino and carboxyl group •For amino acid without ionizable side chains, the Isoelectric Point (equivalence point, pI) is • At this point, the net charge is zero – AA is least soluble in water – AA does not migrate in electric field 2 21 pKpK pI + =
- 19. Ionizable Side Chains Can Show Up in Titration Curves • Ionizable side chains can be also titrated • Titration curves are now more complex • pKa values are discernable if two pKa values are more than two pH units apart Why is the side-chain pK so much higher?
- 20. How to Calculate the pI When the Side-chain is Ionizable? • Identify species that carries a net zero charge • Identify pKa value that defines the acid strength of this zwitterion: (pK2) • Identify pKa value that defines the base strength of this zwitterion: (pKR) • Take the average of these two pKa values
- 21. Peptides and Peptide bonds Peptide bond in a di-peptide “Peptides” are small condensation products of amino acids They are “small” compared to proteins (di, tri, tetra… oligo-)
- 22. Peptide Ends are Not the Same Numbering starts from the amino terminus AA1 AA2 AA3 AA4 AA5
- 23. The Three Letter Code • Naming starts from the N-terminus • Sequence is written as: Ala-Glu-Gly-Lys • Sometimes the one- letter code is used: AEGK
- 24. Peptides: A Variety of Functions • Hormones and pheromones: – insulin (think sugar) – oxytocin (think childbirth) – sex-peptide (think fruit fly mating) • Neuropeptides – substance P (pain mediator) • Antibiotics: – polymyxin B (for Gram - bacteria) – bacitracin (for Gram + bacteria) • Protection, e.g. toxins – amanitin (mushrooms) – conotoxin (cone snails) – chlorotoxin (scorpions)
- 25. Proteins are: • Cofactor is a general term for functional non-amino acid component – Metal ions or organic molecules • Coenzyme is used to designate an organic cofactors – NAD+ in lactate dehydrogenase • Prosthetic groups are covalently attached cofactors – Heme in myoglobin • Polypeptides (covalently linked α-amino acids) + possibly – • cofactors, • coenzymes, • prosthetic groups, • other modifications
- 26. Polypeptide Size in Some Proteins
- 27. Classes of Conjugated Proteins
- 28. Peptides and Proteins- Burning Questions Sequence and composition? Three-dimensional structure? Folding Mechanism? Biochemical role? Functional regulation? Molecular interactions with small and macro-molecules? Structural and sequence relatives? Cellular and sub-cellular localization? Physical and chemical properties?
- 29. Purification – Fractionation of Protein Mixtures • Separation relies on differences in physico- chemical properties – Solubility – Selective Precipitation (Centrifugation) – Thermal stability -- – Charge --Electrophoresis, Isoelectric Focusing, IEC – Size – Dialysis, Sedimentation (Centrifugation), GFC – Affinity for a ligand – “Pull down” assays (Centrifugation), AC – Hydrophobicity (HIC) • Chromatography is commonly used for preparative separation
- 31. Separation by Charge •Ion Exchange Chromatography •Anion exchange Matrix positive Proteins negative Displaced by anions •Cation exchange – Opposite • pH determines net charge on Proteins •Salt concentration gradient •Native gel electrophoresis •Iso-electric Focusing
- 32. Separation by Size • Size exclusion (Gel Filtration) Chromatography – Loading vol. <5% of column volume – Samples diluted • Dialysis or Centrifugal concentrators
- 33. Separation by Affinity • Affinity Chromatography • Free Ligand-Beads -- centrifugation • Ligand-Magnetic- Beads • Immuno-assays on solid supports
- 34. Electrophoresis for Protein Analysis Separation in analytical scale is commonly done by electrophoresis – Electric field pulls proteins according to their charge – Gel matrix hinders mobility of proteins according to their size and shape
- 35. SDS PAGE: Molecular Weight • SDS – sodium dodecyl sulfate – a detergent • SDS micelles binds to, and unfold all the proteins – SDS gives all proteins an uniformly negative charge – The native shape of proteins does not matter – Rate of movement will only depend on size: small proteins will move faster -
- 37. Spectroscopic Detection of Aromatic Amino Acids • The aromatic amino acids absorb light in the UV region • Proteins typically have UV absorbance maxima around 275-280 nm • Tryptophan and tyrosine are the strongest chromophores • Concentration can be determined by UV-visible spectrophotometry using Beers law: A = ε·c·l
- 38. Chapter 3: Summary In this chapter, we learned about: • The many biological functions of peptides and proteins • The structures and names of amino acids found in proteins • The ionization properties of amino acids and peptides • The methods for separation and analysis of proteins
- 39. Nonpolar, Aliphatic R Groups
- 40. Aromatic R Groups Also Hydrophobic These amino acid side chains absorb UV light at 270-280 nm
- 41. Polar, Uncharged R Groups These amino acids side chains can form hydrogen bonding Cysteine can form disulfide bonds
- 42. Basic R Groups
- 43. Acidic R Groups
Editor's Notes
- FIGURE 3-16 Column chromatography. The standard elements of a chromatographic column include a solid, porous material (matrix) supported inside a column, generally made of plastic or glass. A solution, the mobile phase, flows through the matrix, the stationary phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is layered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. The overall protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separation improves (i.e., resolution increases) as the length of the column increases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions.
- In this case the effect of charge is eliminated by binding a negatively charged detergent, SDS, to all the proteins which are denatured. The SDS binds uniformly per unit length of protein and therefore the force on the molecules from the field will be a uniform amount per unit length and the only affect on the speed of travel will be the retarding force due to their size. This is therefore a method to separate molecules based on their molecular weights. Clearly not useful for oligomers since these will be forced apart by the SDS.
- Pehr Edman and Frederick Sanger