09/02/08Biochemistry: Proteins Proteins and Protein Methods Andy Howard Introductory Biochemistry, Fall September 2008

09/02/08 Biochemistry: Proteins Page 2 of 39 Plans for Today pKa’s for main- chain atoms Side-chain Reactivity Acid-base reactivity Other reactions Peptides The peptide bond Main-chain torsion angles , ,  Proteins Protein Purification Salting Out Chromatographic Techniques

09/02/08 Biochemistry: Proteins Page 3 of 39 Why does pK a depend on the side chain? Opportunities for hydrogen bonding or other ionic interactions stabilize some charges more than others More variability in the amino terminus, i.e. the pK a of the carboxylate group doesn’t depend as much on R as the pK a of the amine group

09/02/08 Biochemistry: Proteins Page 4 of 39 How do we relate pK a to percentage ionization? Derivable from Henderson- Hasselbalch equation If pH = pK a, half-ionized One unit below: 90% at more positive charge state, 10% at less + charge state One unit above: 10% / 90%

09/02/08 Biochemistry: Proteins Page 5 of 39 Don’t fall into the trap! Ionization of leucine: pH %+ve % neutral %-ve Main species NH 3 +- CHR- COOH NH 3 + C HR- COO - NH 2 - CHR- COO -

09/02/08 Biochemistry: Proteins Page 6 of 39 Side-chain reactivity Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups Side chains can participate in reactions: Acid-base reactions Other reactions In proteins and peptides, the side-chain reactivity is more important because the main chain is locked up!

09/02/08 Biochemistry: Proteins Page 7 of 39 Acid-base reactivity on side chains Asp, glu: side-chain COO - : Asp sidechain pK a = 3.9 Glu sidechain pK a = 4.1 Lys, arg: side-chain nitrogen: Lys sidechain –NH 3 + pK a = 10.5 Arg sidechain =NH 2 + pK a = 12.5

09/02/08 Biochemistry: Proteins Page 8 of 39 Acid-base reactivity in histidine It’s easy to protonate and deprotonate the imidazole group

09/02/08 Biochemistry: Proteins Page 9 of 39 Cysteine: a special case The sulfur is surprisingly ionizable Within proteins it often remains unionized even at higher pH

09/02/08 Biochemistry: Proteins Page 10 of 39 Ionizing hydroxyls X–O–H  X–O - + H + Tyrosine is easy, ser and thr hard: Tyr pK a = 10.5 Ser, Thr pK a = ~13 Difference due to resonance stabilization of phenolate ion:

09/02/08 Biochemistry: Proteins Page 11 of 39 Resonance-stabilized ion

09/02/08 Biochemistry: Proteins Page 12 of 39 Other side-chain reactions Little activity in hydrophobic amino acids other than van der Waals Sulfurs (especially in cysteines) can be oxidized to sulfates, sulfites, … Nitrogens in his can covalently bond to various ligands Hydroxyls can form ethers, esters Salt bridges (e.g. lys - asp)

09/02/08 Biochemistry: Proteins Page 13 of 39 Phosphorylation ATP donates terminal phosphate to side- chain hydroxyl of ser, thr, tyr: ATP + Ser-OH  ADP + Ser-O-(P) Similar activity adds P to his N Often involved in activating or inactivating enzymes Under careful control of enzymes called kinases and phosphatases

09/02/08 Biochemistry: Proteins Page 14 of 39 Peptides and proteins Peptides are oligomers of amino acids Proteins are polymers Dividing line is a little vague: ~ aa. All are created, both formally and in practice, by stepwise polymerization Water eliminated at each step

09/02/08 Biochemistry: Proteins Page 15 of 39 Growth of oligo- or polypeptide

09/02/08 Biochemistry: Proteins Page 16 of 39 The peptide bond The amide bond between two successive amino acids is known as a peptide bond The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character

09/02/08 Biochemistry: Proteins Page 17 of 39 Double-bond character of peptide

09/02/08 Biochemistry: Proteins Page 18 of 39 The result: planarity! This partial double bond character means the nitrogen is sp 2 hybridized Six atoms must lie in a single plane: First amino acid’s alpha carbon Carbonyl carbon Carbonyl oxygen Second amino acid’s amide nitrogen Amide hydrogen Second amino acid’s alpha carbon

09/02/08 Biochemistry: Proteins Page 19 of 39 Rotations and flexibility Planarity implies  = 180, where  is the rotation angle about N-C bond Free rotations are possible about N- C  and C  -C bonds Define  = rotation about N-C  Define  = rotation about C  -C We can characterize main-chain conformations according to , 

09/02/08 Biochemistry: Proteins Page 20 of 39 Ramachandran angles G.N. Ramachandran

09/02/08 Biochemistry: Proteins Page 21 of 39 Preferred Values of  and  Steric hindrance makes some values unlikely Specific values are characteristic of particular types of secondary structure Most structures with forbidden values of  and  turn out to be errors  generally between -180º and -60º  generally between 30º and 200º or -30 to -80

09/02/08 Biochemistry: Proteins Page 22 of 39 Ramachandran plot Cf. fig. 4.9 in Horton Exceptions are rare except with glycine

09/02/08 Biochemistry: Proteins Page 23 of 39 How to remember  and  Proteins are synthesized N to C on the ribosome Therefore the natural way to draw an amino acid is (NH-CHR-CO)  is the first of those angles  is the second  is earlier in the Greek alphabet, and phi comes before psi in Roman spelling

09/02/08 Biochemistry: Proteins Page 24 of 39 Why bother with mnemonics? Very few textbooks provide memory aids like these You’re grown-ups; you can read the actual answers in your textbook This is intended as a study aid, which is what an instructor should be providing We’ll do several during the semester

09/02/08 Biochemistry: Proteins Page 25 of 39 How are oligo- and polypeptides synthesized? Formation of the peptide linkages occurs in the ribosome under careful enzymatic control (the enzyme is an RNA molecule) Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine): GTP + n-length-peptide + amino acid  GDP + P i + (n+1)-length peptide

09/02/08 Biochemistry: Proteins Page 26 of 39 What happens at the ends? Usually there’s a free amino end and a free carboxyl end: H 3 N + -CHR-CO-(peptide) n -NH-CHR-COO - Cyclic peptides do occur Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step.

09/02/08 Biochemistry: Proteins Page 27 of 39 Reactivity in peptides & proteins Main-chain acid-base reactivity unavailable except on the ends Side-chain reactivity available but with slightly modified pK a s. Terminal main-chain pK a values modified too Environment of protein side chain is often hydrophobic, unlike free amino acid side chain

09/02/08 Biochemistry: Proteins Page 28 of 39 iClicker: What’s the net charge in ELVIS at pH 7? (a) 0 (b) +1 (c) -1 (d) +2 (e) -2 You have 60 seconds here so you can look up the 1-letter codes again!

09/02/08 Biochemistry: Proteins Page 29 of 39 Disulfides In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains

09/02/08 Biochemistry: Proteins Page 30 of 39 What could this do? Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another This can influence protein stability

09/02/08 Biochemistry: Proteins Page 31 of 39 Protein Purification Why do we purify proteins? To get a basic idea of function we need to see a protein in isolation from its environment That necessitates purification An instance of reductionist science Full characterization requires a knowledge of the protein’s action in context

09/02/08 Biochemistry: Proteins Page 32 of 39 Salting Out Most proteins are less soluble in high salt than in low salt In high salt, water molecules are too busy interacting with the primary solute (salt) to pay much attention to the secondary solute (protein) Various proteins differ in the degree to which their solubility disappears as [salt] goes up We can separate proteins by their differential solubility in high salt.

09/02/08 Biochemistry: Proteins Page 33 of 39 How to do it Dissolve protein mixture in highly soluble salt like Li 2 SO 4, (NH 4 ) 2 SO 4, NaCl Increase [salt] until some proteins precipitate and others don’t You may be able to recover both: The supernatant (get rid of salt; move on) The pellet (redissolve, desalt, move on) Typical salt concentrations > 1M

09/02/08 Biochemistry: Proteins Page 34 of 39 Dialysis Some plastics allow molecules to pass through if and only if MW < Cutoff Protein will stay inside bag, smaller proteins will leave Non-protein impurities may leave too.

09/02/08 Biochemistry: Proteins Page 35 of 39 Gel-filtration chromatography Pass a protein solution through a bead- containing medium at low pressure Beads retard small molecules Beads don’t retard bigger molecules Can be used to separate proteins of significantly different sizes Suitable for preparative work

09/02/08 Biochemistry: Proteins Page 36 of 39 Ion-exchange chromatography Charged species affixed to column Phosphonates (-) retard (+)charged proteins: Cation exchange Quaternary ammonium salts (+) retard (-)charged proteins: Anion exchange Separations facilitated by adjusting pH

09/02/08 Biochemistry: Proteins Page 37 of 39 Affinity chromatography Stationary phase contains a species that has specific favorable interaction with the protein we want DNA-binding protein specific to AGCATGCT: bind AGCATGCT to a column, and the protein we want will stick; every other protein falls through Often used to purify antibodies by binding the antigen to the column

09/02/08 Biochemistry: Proteins Page 38 of 39 Metal-ion affinity chromatography Immobilize a metal ion, e.g. Ni, to the column material Proteins with affinity to that metal will stick Wash them off afterward with a ligand with an even higher affinity We can engineer proteins to contain the affinity tag: poly-histidine at N- or C-terminus

09/02/08 Biochemistry: Proteins Page 39 of 39 High-performance liquid chromatography Many LC separations can happen faster and more effectively under high pressure Works for small molecules Protein application is routine too, both for analysis and purification FPLC is a trademark, but it’s used generically