Chapter 3: Amino Acids Polypeptides

1. Chapter 3: Amino Acids & Polypeptides Amino acid: H2N-CHR-CO2H There are 20 different R groups.? thus, 20 different amino acids

2. Zwitterion In aqueous solution, the amino and carboxylic acid groups will ionize to give the zwitterionic form:+H3N-CHR-CO2-

4. Stereochemistry Note that the R group means that the ?-carbon is a chiral center. All natural amino acids are L-amino acids. This means that almost all have the S configuration. (Exceptions: glycine and cysteine � can you tell why?)

15. You should know the structures of the side chains of all 20 natural amino acids both the 1-letter and 3-letter codes (see Table 3-1). the pKa's of the 7 ionizable R groups (only to 1 decimal place) Although you don't have to memorize all of the pKa's for every carboxylate and amine, you should know that they are all ~2 and 9-10, respectively (with a few exceptions). You should should be able to calculate pI if given the pKa's.

19. Can group into several categories: 1) alkanes: A V I L (P) 2) aromatics: F W Y (H) 3) carboxylates: D E 4) corresponding amides: N Q 5) positively charged: K R (H) 6) Sulfur-containing: C M 7) hydroxyls: S T Y

20. Can group into several categories: 8) ?-branched: V T I 9) small: G A S C (V T�) 10) large: W R Y F (M�) 11) H-bond donors: S T Y N Q K R H W (D & E, if protonated) 12) H-bond acceptors: S T Y N Q D E R H W

21. How to calculate pI The isoelectric point (pI) of an amino acid or peptide is the pH at which the charge of the molecule = 0. It can be calculated simply as the arithmetic mean of the 2 pKa's corresponding to the transitions generating the +1 and -1 forms.

22. How to calculate pI Here�s how to do it: Identify all ionizable groups Assign pKa�s to each ionizable group Start with each ionizable group in protonated form (very low pH � maybe 0 or 1) and calculate its net charge Slowly move up in pH to the first ionizable group�s pKa and deprotonate it (reduce charge by 1) Do this until each group is deprotonated. Now you have identified all charged forms and at which pH each transition occurs. Identify the form with net charge = 0 Take the pKa on either side of the electrically neutral form and take their average. This is the pI.

23. How to calculate pI Take Glycine as an example � it has only 2 ionizable groups. The transition (from low to high pH) would be:Gly+1 ? Gly0 ? Gly -1 pKa (-CO2H) = 2.34; pKa (-NH3+) = 9.60 pI =(2.34 + 9.60)/2 = 11.94/2 = 5.97

25. How to calculate pI Glutamate has an ionizable group (-CO2H; pKa = 4.25) that generates a negative charge when deprotonated.Its transitions would be: Glu+1 ? Glu0 ? Glu-1 ? Glu-2 The relevant pKa 's are pKa(-CO2H) = 2.19; pKa(R) = 4.25 pI =(2.19 + 4.25)/2 = 6.44/2 = 3.22

27. How to calculate pI Histidine has an ionizable group (imidazole; pKa = 6.00) that is positively charged when protonated. Its transitions would be: His+2 ? His+1 ? His0 ? His-1 The relevant pKa 's are pKa(R) = 6.00; pKa(-NH3+) = 9.17 pI =(6.00 + 9.17)/2 = 15.17/2 = 7.59

29. Making dipeptides +H3N-CHR-CO2- + +H3N-CHR-CO2- ? +H3N-CHR-CONH-CHR-CO2- + H2O This process can be repeated to make a tripeptide and so on: +H3N-CHR-CONH-CHR-CO2- + +H3N-CHR-CO2- ? +H3N-CHR-CONH-CHR-CONH-CHR-CO2-

32. Making dipeptides The C-N bond has partial double bond character, making the -CONH- moiety planar. This limits the orientations available to the polypeptide (e.g. the barrier of rotation about the C-N bond in formamide is ~18 kcal/mol)

36. Hydrolysis of polypeptides& amino acid analysis Polypeptides can be hydrolyzed to constituent amino acids. This is typically done by boiling the polypeptide in 6 M HCl for 24 hours. +H3N-CHR-CONH-CHR-CONH-CHR-CO2- + 2 H2O ? 3 +H3N-CHR-CO2-

37. Hydrolysis of polypeptides& amino acid analysis The R groups remain intact, except for: Trp � indole ring damaged Asn, Gln � converted to Asp, Glu

38. Hydrolysis of polypeptides& amino acid analysis The amino acids can be derivatized with o-phthalaldehyde to make fluorescent derivatives that are easy to detect. These are chromatographed by reverse-phase HPLC (high-pressure liquid chromatography). The characteristic retention times are used to identify the amino acids. The fluorescence level can be quantified to determine the amount of that amino acid.

39. Amino acid analysis.

40. Disulfide bonds 2 cysteine ? cystine 2 R-SH ? R-S-S-R (Note: This is an oxidation) Intracellular conditions are maintained sufficiently reducing to inhibit formation of most disulfide bonds. Extracellular conditions (as well as those found in some organelles) are more oxidizing, favoring disulfide formation. Thus, extracellular proteins containing cysteines often have disulfides, while intracellular (cytosolic) proteins rarely have disulfides.

42. Reactions with amino acids: I. Amino group Acylation ? R-(C=O)-NH-R� Ninhydrin reactionCauses oxidative decarboxylation of ?-amino acids, and release of ammonia, which reacts with a second molecule of ninhydrin to form a purple product.(You don�t need to know details � just know that it reacts with any free amino group and the final product is purple.)

43. Reactions with amino acids: I. Amino group Ninhydrin reaction

44. Reactions with amino acids: Fluorodinitrobenzene reaction Dansyl chloride reaction

45. Reactions with amino acids: Fluorescamine reactionSimilar to dansyl chloride � forms fluorescent adduct.(Don�t need to know details.)

46. o-phthalaldehyde reaction Schiff's base formation R-HC=O + NH2-R' ? R-HC=N-R' + H2O 8. Edman degradation (more later) Reactions with amino acids:

47. Reactions with amino acids: Carboxyl group Amide formation  Ester formation Acyl halide formation Reduction to alcohol (via aldehyde)

48. Reactions with amino acids: Side chains R-SH (cysteine) & R-S-S-R (cystine) Reduction of disulfide with ?-mercaptoethanolR-S-S-R' + HS-CH2CH2OH ?? R-S-S-CH2CH2OH + R'-SHR-S-S-CH2CH2OH + HS-CH2CH2OH ? HOCH2CH2-S-S-CH2CH2OH + R-SH(driven by mass action) Can also accomplish with dithiothreitol (DTT) (only requires 1 molecule to reduce 1 disulfide)

49. Coupling to N-ethylmaleimide(blocks disulfide formation after reduction) Carboxymethylation with iodoacetate (introduces carboxylate)  R-SH + I-CH2-COOH ? R-S-CH2-COOH + HI Reactions with amino acids:

50. Reactions with amino acids: Reaction with ethyleneimine (introduces amino group) 5. Performic acid oxidation to cysteic acid 3 HCOOOH + R-SH ? R-SO3H + 3 HCOOH 5 HCOOOH + R-S-S-R + H2O ?  2 R-SO3H + 5 CHOOH 6. Reaction with mercurials R-SH + R'-Hg-Cl ? R-S-Hg-R' + HCl

51. Reactions with amino acids: B. Imidazole (histidine) 1. Acetylation (with IAA) C. Phenol (tyrosine) Nitration

52. Reactions with amino acids: Acetylation iodination (important use: isotopic labeling with 125I)

53. Reactions with amino acids: D. ?-NH2 (lysine) Acylation R-NH-R� Fluorodinitrobenzene reaction Dansyl chloride reaction o-phthalaldehyde reaction Schiff's base formation cyanylationR-NH2 + HNCO ? R-NH-CONH2 amidination

54. Reactions with amino acids: R-COOH (glutamate, aspartate) Amidation Esterification Acyl halide formation Reduction to alcohol R-CONH2 (glutamine, asparagine) DeamidationR-CONH2 + H2O ? R-COOH + NH3

55. Protein Sequencing Strategy 1) Purify protein (methods discussed later) Cleave disulfides � react with: reducing agent followed by alkylating agent DTT or ?-ME NEM or IAA performic acid

57. Protein Sequencing Strategy Determine ends N-terminus � react with FDNB or dansyl chloride followed by acid hydrolysis and HPLC(Can also perform Edman degradation with intact protein to get N-terminal sequence, if the N-terminus is not blocked.) C-terminus � digest with carboxypeptidase(not often done)

58. Protein Sequencing Strategy Cleave polypeptide into smaller peptides A) Endopeptidases trypsin � cleaves after Lys and Arg chymotrypsin � cleaves after Phe, Tyr, or Trp endoproteinase Glu-C � cleaves after Glu

60. Protein Sequencing Strategy Cleave polypeptide into smaller peptides B) Chemicals CNBr � cleaves after Met(Lactone will be hydrolyzed during acid hydrolysis.)

61. Protein Sequencing Strategy Purify peptides Characterize peptides(i.e. spectroscopy, amino acid analysis, chromatography, etc.) Most important � determine amino acid sequence by Edman degradation

63. Protein Sequencing Strategy 7) Reassemble sequence through overlaps of peptides created by different means 8) Map disulfides by cleaving protein into peptides before disulfide bond cleavage. After purification of disulfide-linked peptides and cleavage of their disulfide bonds, sequencing of the peptides should reveal which cysteines are linked in disulfide bonds.

64. Edman degradation Q: Why can you not use it to sequence long polypeptides? A: Each step is completed with <100% yield. Eventually, less than half of the peptides have the "current" amino terminal amino acid residue.

65. Example (inefficiency exagerated): H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-� After 1st step� H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-V-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-� After 2nd step� H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-�H2N-D-R-G-T-H-K-L-S-F-E-W-Q-C-V-N-� And so on�

67. Synthesis of polypeptide Artificial (Merrifield process): Utilizes amino acid coupled to a solid phase support through the carboxylate. Amino acids with protected amino groups and activated carboxylates react with the amino acid to make N-terminally protected dipeptide. After removal of the protecting group, another protected and activated amino acid can be added to the N-terminus. The cycle is repeated until the desired polypeptide has been synthesized. It can then be removed from the solid support by treatment with strong acid. (This also removes the protecting groups on the amino acid sidechains that prevent unwanted reactions.)

73. Natural polypeptide synthesis Performed on the ribosome, a large aggregate of RNA and protein; in effect, "solid phase". Protecting groups are not necessary, as the sequence is determined by genetic programming. The C-terminus is activated by coupling to a nucleic acid (tRNA). This tRNA serves both as the activating group and the connection to the ribosome. The polypeptide is synthesized starting from the N-terminal amino acid.

81. Protein Purification In an organism, any one protein is present as a very small percentage of the total biomolecules. In order to characterize a protein fully, it is necessary to purify it. There are many possible strategies that one could use to purify a protein. In general, the more that you know about the protein's properties, the better strategy you could design to purify it. A typical protein purification strategy will be comprised of several stages, each one taking advantage of different characteristics of the protein.

82. Modulating Solubility 1) Precipitation at the pI A protein's average net charge at its isoelectric point is 0. Above or below this pH, the protein molecules are negatively or positively charged, respectively, causing them to repel each other.

83. Modulating Solubility 2) Salting in Many proteins are poorly soluble in pure water, but are much more soluble in salt-containing solutions. Thus, lowering ionic strength can be used to precipitate certain proteins.

84. Solubility of lactoglobin as a function of pH at several NaCl concentrations.

85. Modulating Solubility 3) Salting out At very high concentrations (>1 M) of certain salts, proteins solubility is reduced due to a competition with the protein for interaction with water molecules.

86. Solubility of hemoglobin at its pI as a function of ionic strength and ion type

87. Ammonium sulfate precipitation Precipitation with ammonium sulfate is a common first step in protein purification. Because proteins precipitate at different concentrations of salt, one can perform a first "cut" with a concentration that will leave the desired protein soluble, remove the precipitate, and then add sufficient salt to precipitate the desired protein.

88. Solubilities of several proteins in ammonium sulfate solutions.

89. Ammonium sulfate precipitation Example: Your protein will remain soluble at 30% (w/v) ammonium sulfate, but precipitates at 40% ammonium sulfate. You slowly add the salt to your protein extract until it reaches a concentration of 30%, allow precipitation to occur, and then centrifuge the solution to remove the precipitate. You take the supernatant, and add ammonium sulfate until it reaches a concentration of 40%, allow precipitation to occur, and then centrifuge the solution to remove the precipitate. You dissolve the precipitate in a buffer and dialyze it to remove the salt.

90. Dialysis The protein is put in a bag of cellulose membranes having small pores of controlled size. Proteins bigger than the pores are retained, while smaller molecules may diffuse out. As the volume of the buffer surrounding the bag is many times (100-1000x) the volume within the bag, the smaller molecules can be effectively removed after several changes of the outer buffer.

91. Dialysis

92. Gel filtration chromatography(a.k.a. "molecular sieve", "size exclusion") The protein is applied to the top of a column consisting of porous beads made of a hydrated material, such as agarose, dextran, or polyacrylamide. The pores of the beads are of a controlled size and this regulates which proteins can enter the beads. The larger proteins will be excluded from the beads and will flow through the column faster than the smaller molecules, which experience a much larger volume. In practice, there exists a range of pore sizes, and proteins are separated by their sizes (and shapes): the largest are eluted first, and the smallest elute last.

93. Gel filtration chromatography

94. Gel filtration chromatography Vbed = Vbeads + Vvoid The void volume is the volume surrounding the beads. The bed volume is the total volume of the column. Vvoid is typically about Vbed /3 A protein can be characterized by its elution volume (Velution), which is the volume of solvent required to elute it from the column. The relative elution volume (= Velution / Vvoid) is a quantity independent of the particular column used. By standardizing the column with proteins of known size, one can use this technique to estimate molecular weight (assuming that the shape is close to that of the standards � more or less spherical).

96. Commonly Used Gel Filtration Materials

97. Ion exchange chromatography This technique uses the charge on a protein to separate it from other proteins. In the process of ion exchange, ions in solution replace ions that are electrostatically bound to an inert support carrying groups with the opposite charge. Cation exchangers bear negatively charged groups. Anion exchangers bear positively charged groups. Polyelectrolytes, such as proteins, can bind to either cation or anion exchangers, depending on their net charge (i.e. depending on the pH).

98. Ion exchange chromatography In most cases, a column is prepared with the ion exchanger, which is then equilibrated with the same buffer used to dissolve the protein. The exchanger and pH are chosen such that the protein will bind relatively tightly to the ion exchanger. The protein solution is loaded to the top and the column is washed with the buffer, removing all proteins with the opposite charge or low charge. The protein can be eluted from the column either by changing the pH or by increasing the salt concentration, which shields the charges and thus decreases their attraction. Elution is most often carried out by applying a salt gradient to the column.

100. Ion exchange chromatographyusing stepwise elution.

101. Device for generating a linear concentration gradient.

102. Molecular formulas of cellulose-based ion exchangers.

103. Some common ion exchangers.

104. Affinity chromatography This technique takes advantage of the fact that many proteins specifically bind other molecules as part of their function. One can use this information to construct a column containing the ligand covalently attached to a matrix. Upon passing the protein solution through such a column, only the proteins that can bind the ligand will be retained on the column. Then the conditions can be adjusted to effect release from the ligand. Often this can be done simply by eluting with the soluble version of the ligand.

106. Covalent linking of ligand to agarose

107. Derivatization of epoxy-activated agarose.

108. Example: purification of staphylococcal nuclease by affinity chromatography on bisphosphothymidine-linked agarose

109. Preparative vs. Analytical Preparative methods used to purify proteins Able to handle large amounts of protein at once The chromatographic techniques just discussed are all preparative. Analytical methods used to analyze proteins Usually deal with small amounts of protein Some preparative techniques can also have analytical formats.

110. Analytical techniques Separation based on Mass Charge Shape Different combinations of the above

111. Density Gradients An equilibrium sedimentation experiment can be set up with linear gradients of sucrose or glycerol. The protein is loaded on top, and centrifugation is started. The advantage of this technique is that the gradients tend to be rather stable and allow one to remove the protein of interest from them. The disadvantage is that they are not so accurate for determination of S. Often such gradients are run with molecular weight standards of known molecular weights to allow estimation of molecular weight. The sedimentation coefficient of a protein is approximately proportional to the 2/3 power of its molecular weight. Note that this only holds true for proteins with a globular shape (i.e. quasi-spherical).Sunknown/Sstandard � (Munknown/Mstandard)2/3

112. Zonal ultracentrifugation

113. Zonal ultracentrifugation

114. Gel electrophoresis General technique to analyze mixtures of proteins, and for limited purification of proteins. The protein is driven through a viscous solvent by an applied electric field (E), due to the charge of the protein (z): v = Ez/f (f = the protein's frictional coefficient) A protein�s electrophoretic mobility (�) is defined: � = v/E Typically this is performed in the presence of a gel support, such as polyacrylamide prevents convection currents enhances separation by serving as a molecular sieve

115. Polymerization of acrylamide and N,N�-methylenebisacrylamide

116. Native gel electrophoresis The native protein migrates through the gel according to the charge of the protein at the pH of the buffer system. Useful for getting relatively pure protein, but in small amounts. Not used very often.

117. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) Most common form of PAGE, but not useful for purification of proteins in their native conformation. Proteins are solubilized with the detergent SDS (sodium dodecyl sulfate): binds polypeptide at a ratio of ~1 SDS per 2 amino acid residues denatures protein ? converts to roughly rod-like shape protein has a negative charge roughly proportional to its mass The additional negative charge is much greater than the protein's intrinsic charge, which can usually be ignored.

118. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) As charge/mass ratio is almost constant and the molecular shapes are all similar, separation is on the basis of size. Smaller polypeptides migrate faster and larger ones migrate slower, due to the gel filtration effect. There is an empirical relationship between mobility and molecular weight: � ? 1/log Mr Average pore size of the gel can be controlled by varying the concentration of acrylamide before initiating polymerization. Higher percentage polyacrylamide gels (smaller pore sizes) will result in better resolution of smaller polypeptides.

120. slab gel electrophoresis

124. Isoelectric focusing A pH gradient is set up by electrophoresing polyampholytes (300-600 Da oligomers bearing amino and carboxylate groups in varying rations) in a gel tube. The more basic ones (cationic) will accumulate near the cathode and the more acidic (anionic) will accumulate near the anode, thereby establishing a continuous pH gradient. When a protein is applied to the gel, it will migrate toward the anode if it is negatively charged or toward the cathode if positively charged, until it reaches the region corresponding to its pI, where it will stop. Proteins are often denatured in 6M urea, which does not change the protein's charge (unlike SDS).

125. General formula of the ampholytes used in isoelectric focusing.

126. Example A protein with a pI of 5.2 is introduced near the cathode end, which has pH of 9.5 (in this gel). It is then negatively charged and will migrate toward the anode. As it migrates in this direction, the pH will steadily decrease, and the amount of negative charge carried by the protein will also decrease, as more and more ionizable groups become protonated. Thus it will slow down, until it hits the region where pH = 5.2, where it will experience no further force. If it diffuses in either direction, it will pick up charge and the electric field will force it back. This results in the protein being concentrated (focused) in a very narrow region of the gel.

129. 2-dimensional gels This is a powerful technique that combines isoelectric focusing with SDS-PAGE. Proteins are separated in the first dimension by isoelectric focusing (IEF). Then this tube is attached to the side of an SDS-polyacrylamide gel. SDS-PAGE provides the second dimension. Each protein migrates to a semi-unique spot according to its pI and molecular weight (MW).

132. Two-dimensional (2D) gel electrophoresis

133. Protein Purification: Practical Aspects All cells contain proteases � enzymes that catalyze hydrolysis of peptide bonds. Upon breaking cells, these are released into the extract, where they can degrade the protein you want to purify. In order to inhibit proteolysis and denaturation, protein purification is usually carried out in the cold (on ice or in a cold room) in the presence of protease inhibitors (small molecules that inhibit specific proteases).

134. Protein Purification: Practical Aspects You often have to choose your source of material carefully unicellular organism organ of a metazoan (or plant)? Where is the enzyme located? In cytosol? Within membrane-bound organelle? Secreted? You can use differential centrifugation to do a (crude) separation of various cellular compartments to get a starting point. If the protein is in a specific organelle, density gradient centrifugation is usually used to purify the organelle first.

139. Purification table

140. Purification table Yield and purification factor can be expressed for the individual step OR as cumulative yield or purification factor, taking into account all steps up to to that point You need to be careful to distinguish between them.

142. Modern tricks: Genetic engineering assists protein purification In the past, one often had to purify a protein in order to obtain enough sequence information to search for the gene. Nowadays, one often has a gene encoding a protein long before the protein has been purified. In either case, "engineering" the gene can ease protein purification.

143. Modern tricks: One can use strong promoters (DNA elements that direct RNA polymerase to transcribe the gene) to overproduce the protein, either in a homologous system, or in a very different system. This increases the specific activity of your starting material. One can attach sequence elements to the gene, such that the protein will have additional polypeptide segments that can be used as "tags" for affinity purification. Example: The sequence H-H-H-H-H-H (hexahistidine) is capable of binding Ni2+ ions. Attachment of the "His6-tag" to a protein allows it to bind to a nickel-nitrilotriacetic acid (NTA-Ni2+) column and be eluted with imidazole.

144. Example: Engineering removable affinity tag Clone gene into plasmid to express in bacteria Fuse protein to intein and chitin-binding domain (CBD) Makes chimeric protein Pass through chitin column and wash off all other proteins. DTT allows intein to cleave link between itself and protein. Protein elutes, leaving intein-CBD on the column.