Combinatorial Biochemistry and Phage Display

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1 Combinatorial Biochemistry and Phage Display Prof. Valery A. Petrenko Director - Valery Petrenko Instructors Galina Kouzmitcheva and I-Hsuan Chen Auburn 2006, Spring semester

2 COMBINATORIAL BIOCHEMISTRY AND PHAGE DISPLAY Lecture 2 Chemical Foundations of Molecular Recognition. Protein structure Valery Petrenko Auburn 2006, Spring semester

3 Chemical principles that govern the recognition properties of biological molecules The covalent bonding of carbon with itself and with other elements The functional groups that occur in common biological molecules The three-dimensional structure and stereochemistry of carbon compounds The effects of chemical structure on reactivity Role of water in biochemical processes Noncovalent bonds

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5 Covalent Bonds Formed when two different atoms share electrons in the outer atomic orbitals Each atom can make a characteristic number of bonds (e.g., carbon is able to form 4 covalent bonds) Covalent bonds in biological systems are typically single (one shared electron pair) or double (two shared electron pairs) bonds

6 Carbon can form single bonds with hydrogen atoms, and both single and double bonds with oxygen and nitrogen atoms. Two carbon atoms can share one, two or three electron pairs, thus forming single, double and triple bonds.

7 The making or breaking of covalent bonds involves large energy changes In comparison, thermal energy at 25ºC is < 1 kcal/mol

8 Covalent bonds have characteristic geometries Tetrahedral

9 Covalent double bonds cause all atoms to lie in the same plane

10 Configuration - the fixed spatial arrangement of atoms in an organic molecule that is conferred by the presence of 1. double bonds around which there is no freedom of rotation, or 2. chiral centers around which substituent groups are arranged in a specific sequence Geometric or cis-trans isomers

11 Carbon atoms that are bound to four different atoms or groups are said to be asymmetric The bonds formed by an asymmetric carbon can be arranged in two mirror images -stereoisomers Stereoisomers are either right-handed handed or left- handed and have different biological activities Asymmetric carbons are key features of amino acids and carbohydrates Enantiomers mirror images Diastereomers stereoisomers which are not mirror images

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13 Conformation spatial arrangement of substituent groups that, without breaking any bonds, are free to assume different positions in space because of the freedom of bond rotation.

14 Electrons are shared unequally in polar covalent bonds Atoms with higher electronegativity values have a greater attraction for electrons

15 The hydrogen bond underlies water s s chemical and biological properties A water molecule has a net dipole moment caused by unequal sharing of electrons Molecules with polar bonds that form hydrogen bonds with water can dissolve in water and are termed hydrophilic

16 Biological fluids have characteristic ph values Aqueous solutions contain some concentration of H + and OH - ions, the dissociation products of water In pure water, [H + ] = [OH - ] = 10-7 M The concentration of H + in a solution is expressed as ph ph = -log [H + ] For pure water, ph = 7.0 On the ph scale, 7.0 is neutral, ph < 7.0 is acidic, and ph > 7.0 is basic The cytosol of most cells has a ph of 7.2

17 Hydrogen ions are released by acids and taken up by bases When acid is added to a solution, [H + ] increases and [OH - ] decreases When base is added to a solution, [H + ] decreases and [OH - ] increases The degree to which an acid releases H + or a base takes up H + depends on the ph

18 The Henderson-Hasselbalch Hasselbalch equation The Henderson-Hasselbalch Hasselbalch equation relates the ph and K eq of an acid-base system ph = pk a + log [A- ] [HA] The pk a of any acid is equal to the ph at which half the molecules are dissociated and half are neutral (undissociated( undissociated) It is possible to calculate the degree of dissociation if both the t ph and the pk a are known

19 Buffers ensure that ph remains relatively constant The titration curve for phosphoric acid (H 3 PO 4 ), a physiologically important buffer

20 Noncovalent bonds Several types: hydrogen bonds, ionic bonds, van der Waals interactions, hydrophobic bonds Noncovalent bonds require less energy to break than covalent bonds The energy required to break noncovalent bonds is only slightly greater than the average kinetic energy of molecules at room temperature Noncovalent bonds are required for maintaining the three- dimensional structure of many macromolecules and for stabilizing specific associations between macromolecules

21 Multiple weak bonds stabilize large molecule interactions Pauling & Delbruck (1940): Van der Waals interactions, electrostatic interactions, and hydrogen bonds. Kauzmann (1959): hydrophobic effect, an entripy-based attraction

22 Hydrogen bonds within proteins

23 Ionic Bonds The atoms that form the bond have very different electronegativity values and the electron is completely transferred to the more electronegative atom Ionic bonds result from the attraction of a positively charged ion i (cation)) for a negatively charged ion (anion) Ions in aqueous solutions are surrounded by water molecules, which interact via the end of the water dipole carrying the opposite charge of the ion

24 van der Waals interactions are caused by transient dipoles When any two atoms approach each other closely, a weak nonspecific attractive force (the van der Waals force) is created due to momentary random fluctuations that produce a transient electric dipole Van Der Waals radii describe the space-filling dimentions of atoms. When two atoms are joined covalently, the atomic radii at the point of bonding are less than the van der Waals radii

25 Hydrophobic bonds cause nonpolar molecules to adhere to one another Nonpolar molecules (e.g., hydrocarbons) are insoluble in water and are termed hydrophobic Since these molecules cannot form hydrogen bonds with water, it is energetically favorable for such molecules to interact with other hydrophobic molecules This force that causes hydrophobic molecules to interact is termed a hydrophobic bond

26 Multiple noncovalent bonds can confer binding specificity

27 Phospholipids are amphipathic molecules

28 Phospholipids spontaneously assemble via multiple noncovalent interactions to form different structures in aqueous solutions

29 COMBINATORIAL BIOCHEMISTRY AND PHAGE DISPLAY Protein structure Valery Petrenko Auburn 2006, Spring semester

30 Protein structure determines function Proteins are single, unbranched chains of amino acid monomers There are 20 different amino acids A protein s amino acid sequence determines its three-dimensional structure (conformation) In turn, a protein s structure determines the function of that protein

31 All amino acids have the same general structure but the side chain (R group) of each is different Chiral center: cannot be superimposed onto its mirror image

32 Stereoisomers of the amino acid alanine = = = Ball-and-stick model Space filling model

33 Acidic Basic AMINO ACIDS Nonpolar hydrophobic Polar hydrophilic

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36 Peptide bonds connect amino acids into linear chains

37 Four levels of structure determine the shape of proteins Primary: the linear sequence of amino acids Secondary: the localized organization of parts of a polypeptide chain (e.g., the α helix or β sheet) Tertiary: the overall, three-dimensional arrangement of the polypeptide chain Quaternary: the association of two or more polypeptides into a multi-subunit complex

38 Peptide bond is rigid and planar A resonance or partial sharing of two pairs of electrones between the carbonyl oxygen and the amide nitrogen. Atoms associated with the peptide bond are coplanar. The carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, setting up a small electric dipole

39 The peptide C-N bonds are unable to rotate freely because of their partial double-bond character. Rotation is permitted about the N-Cα and Cα-C bonds The bond angles resulting from rotations at Cα are labeled φ (phi) for N-Ca bond and ψ (psi) for the Cα-C bond.

40 Ramachandran plot for L-Ala residues Allowed (no steric interference) Not allowed φ and ψ are both defined as 0 o when the two peptide bonds flanking the a carbon atom are in the same plane

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42 Ramachandran plots and real data The values of φ and ψ for all the amino acid residues except Gly in purivate kinase are overlaid on the plot of theoretically allowed conformations

43 The α helix is stabilized by a hydrogen bond between the H atom attached to the electronegative N atom of a peptide linkage and the electronegative carbonyl O atom of the fourth amino acid on the amino-terminal side of the peptide bond

44 Constraints effecting stability of an α-helix: the electrostatic repultion (or attraction) between successive amino acid residues with charged R groups the bulkiness of ajacent R groups the interaction between amino acid side chains spaced three (or four) residues apart the occurrence of Pro or Gly residues the interaction between amino acid residues at the ends of the helical segment and the electric dipole inherent to the a-helix Asp 100 (red) and Arg 103 (blue) in troponin C

45 Secondary structure: the beta sheet

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47 Primary, secondary, tertiary and quaternary structure in hemagglutinin structure in hemagglutinin

48 Different graphical representations of the same protein

49 Motifs are regular combinations of secondary structures A coiled coil motif is formed by two or more helices wound around one another

50 Other examples of motifs

51 Structural and functional domains are modules of tertiary structure

52 Folding, modification, and degradation of proteins A newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein All molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule

53 The information for protein folding is encoded in the sequence Native state; catalytically active ribonuclease Unfolded state; inactive Native, catalytically active state Christian Anfinsen, 1950s

54 Folding of proteins in vivo is promoted by chaperones