Amino Acids and Protein Intro

1. Amino Acids

6. The 5 categories of amino acids: nonpolar, aromatic, polar, positively charged and negatively charged

7. The 5 categories of amino acids: nonpolar, aromatic, polar, positively charged and negatively charged

9. Proline -Has a distinctive ring structure. -Allows for less flexibility in polypeptide regions containing proline as the secondary imino group of proline is held in a more rigid conformation. -Found often in bends of folded protein chains.

10. Nonstandard amino acids All nonstandard amino acids are derivatives of standard amino acids About 300 nonstandard amino acids have been found. Found in prothrombin Involves 4 lysine residues, Found in elastin Contains selenium instead of sulfur, rare Found in plant cell wall, collagen Derivatives of lysine, found in collagen and myosin, respectively In addition, amino acids can be phosphorylated (thr, tyr, ser) or otherwise modified (cysteinylation. methylation) to modify the function of a protein

12. Titration curves of amino acids; prediction of the electric charge of amino acids Titration curve of 0.1M glycine at 25 °C. Blue boxes show the regions of greatest buffering power Inflection point Inflection point Inflection point A quantitative measure of the pKa of the amino acid Note there are 2 regions of buffering Not a good buffer at Physiological pH Isoelectric Point: pI, the Characteristic pH at which The net charge of a Compound is 0.

14. Amino acids with an ionizable R-group have multiple stages corresponding to the multiple possible ionization steps. 3 pKa values The additional stage for the titration of the ionizable R-group merges to some extent with the other two.

16. The Hendersson-Hasselbach equation also applies to the amino acid acid-base reactions: 2. What is the ratio of forms of glycine when the pH = 10? 1. What is the pH at which the ratio of is 0.25? to to

18. The absorbance of UV light by tryptophan (W) and tyrosine (Y) is exploited in the measurement of amino acids and peptides.

21. Amino Acids and Peptides

24. Acid-Base properties of peptides 1. Isoelectric points of peptides - Peptides have characteristic titration curves and a characteristic isoelectric point (pI) at which their overall charge is neutral and at which they do not move in an electric field 2. peptides can be distinguished by their ionization behavior 3. Acid-Base properties of peptides - The α -amino and carboxyl groups of all non-terminal amino acids are covalently bound in peptide bonds, which do not ionize and therefore do not contribute to the total acid-base behavior of peptides -The R groups of some amino acids can ionize, and in a peptide these R groups contribute to the overall acid/base properties. -The pKa value for an ionizable R group can change when an amino acid becomes a residue in a peptide, due to a change in its surroundings

25. Peptide bonds connect amino acids to form polypeptides and proteins. -The peptide bond is rigid and planar -One exception is the cis-configuration Of peptide bonds involving proline.

26. -3 bonds separate the sequential α -carbons in a peptide chain. The N - C α and C – C α bonds are able to rotate around bond angles designated Φ and Ψ respectively. -The C – N bond is not free to rotate, and other bonds may be sterically hindered depending on the R-group.

27. The conformations of peptides are defined by the values of phi( Φ, degree of rotation at the N- α C bond) and psi( Ψ, degree of rotation at the α -C bond). Conformations deemed possible are those that involve little or no steric interference, based on calculations using known van der waals radii and bond angles. Allowed values for Φ and Ψ are graphically revealed in a Ramachandran plot.

28. Frederick Sanger

29. Structure of bovine insulin – contains 3 disulfide bonds Cystine

30. Sanger’s sequencing method and the (Pehr) Edman degradation method The Edman degradation removes only the amino-terminal amino acid from a polyeptide, leaving all other bonds intact. This process has been automated using a machine called a sequenator .

31. -Hydrolases

32. -Deduction of sequence

34. Protein Structure I and II

36. Proteins

38. -Proteins can be multi-subunit with more than one polypeptide associated non-covalently. -Subunits can be same, or different.

39. Proteins can be identified by their sequence

40. Some proteins have chemical groups other than amino acids -The conjugated group is called the prosthetic group .

42. General principle of column chromatography

43. Types of column chromatography Ion exchange chromatography: Cation or anion exchange The column matrix is a synthetic polymer containing bound charged groups. The affinity of each protein for the charged groups on the column is affected by -pH (ionization state) -Concentration of salt Size exclusion: separates by size. -Matrix is a cross linked polymer of defined size -Larger protein migrates faster as they are too large to enter pores

44. Types of column chromatography (cont.) Affinity: Separates by specifically binding a ligand (such as an antibody) cross-linked to the column

46. Electrophoresis (used for detection and analysis) : -Separates proteins by size, charge and shape. -SDS allows estimation of molecular weight. CH 3 -(CH 2 ) 11 -O-SO 3 - Na +

47. Estimating the molecular weight of a protein Most common is the use of marker proteins of known molecular weight A plot of the log M r of the marker proteins, which allows the estimation of the molecular weight of the unknown protein from the graph.

48. Overview of polyacrylamide gel electrophoresis (PAGE)

49. Overview of Western Blotting

51. Isoelectric Focusing 2D Electrophoresis (cont.) -2. PAGE is then performed on the focused samples. Picture of a 2-dimensional gel

53. Levels of Structure in Proteins Sequence -peptide and disulfide bonds A stable arrangement Folding Arrangement of multimers -Spatial arrangement of atoms in a protein is called conformation -The conformation of a protein under a given condition is thermodynamically stable -Proteins in their functional folded conformation are called native proteins - Stability of a protein is defined as their tendency to maintain a native conformation

54. Secondary structure refers to the arrangement of adjacent amino acids in regular, recurring patterns; a few types of secondary structure are particularly stable and occur widely in proteins: The α helix, β conformation, and β turns The peptide bond is rigid but other bonds are free to rotate. α helix makes optimal use of H-bonds and forms readily. In α helices, the properties of the side chains place five types of constraints on the stability of the helix a. The electrostatic repulsion (or attraction) between successive amino acid residues with charged R groups. For example long block of Glu residue will not form helix as the COO- groups will repel each other and overcome influence of hydrogen bond. The positive charge on Lys and Arg residue would do the same. b. The bulkiness of adjacent R groups. Such as Asn, Ser, Thr and Leu c. The interactions between amino acid side chains spaced three (or four) residues apart. d. The occurrence of Pro and Gly residues. The N atom of Pro is rigid and cannot rotate. Also, it cannot form hydrogen bond as it does not have H. Gly has more conformational flexibility and tends to form a coil.

55. e. The interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the a helix. Often the negatively charged amino acids reside near the N-terminus where they stabilize the interaction with positive charge. Positively charged residue near the N-terminus would destabilize the helix. *Right-handed alpha helix

56. The β Conformation of Polypeptide Chains 1. The backbone of the polypeptide chain is extended into a zigzag rather than a helical structure 2. The zigzag chains can be arranged side by side to form a structure resembling a series of pleats, called a β-sheet 3. H-bonds are formed between adjacent segments of polypeptide chain 4. the adjacent polypeptide chains can be either parallel or antiparallel

57. β turns are common in proteins 1. In globular proteins, which have a compact folded structure, nearly one third of the amino acid residues are in turns or loops where the polypeptide chain reverses direction 2. These are the connecting elements that link successive runs of a helix or β conformation 3. Particularly common are β turns that connect the ends of two adjacent segments of an antiparallel β sheet

60. In a multi-subunit protein, the arrangement of the subunits in 3-D complexes constitutes quaternary structure Quaternary structure of deoxyhemoglobin: 2 α subunits and 2 β subunits pack together to make a multimer of 4 subunits.

61. The light and heavy chains of an antibody molecule.

63. α -Keratin -Alpha keratins belong to the intermediate filament (IF) protein family. -An all α -helix protein. -Rich in hydrophobic amino acids: Ala, Val, Leu, Ile, Met, Phe

64. Collagen -A repeating tripeptide: Gly-X-Pro or Gly-X-Hyp -Left-handed helical structure: 3 residues per turn -3 helices wrap around each other in a right- handed twist -Great tensile strength!

69. - Polypeptide segments adjacent to each other in the primary sequence are usually stacked adjacent to each other in the folded structure. - Connections between elements of secondary structure cannot cross or form knots - β conformations are more stable when the segments are slightly right handed twist Other common motifs (mostly functional) found in many proteins. -Supersecndary structures (motifs): particularly stable arrangements of several Elements of 2° structure and the connections between them.

78. Viral Capsid Protein Polio virus Icosahedron Tobacco Mosaic Virus Helical symmetry

81. X-ray diffraction patterns from whale myoglobin are used to extract diffraction patterns to determine the 3-D structure of the protein Three dimensional electron density map calculated from the diffraction patterns

82. The use of 2D nuclear magnetic resonance (NMR) to determine the 3D structure of whale myoglobin

83. http://www.portfolio.mvm.ed.ac.uk/studentwebs/session2/group5/introliz.htm