1. 3 | Amino Acids, Peptides, and Proteins
© 2017 W. H. Freeman and Company

2. CHAPTER 3 Amino Acids, Peptides, and Proteins
Learning goals:
Structure and naming of amino acids
Structure and properties of peptides
Ionization behavior of amino acids and peptides
Methods to characterize peptides and proteins

3. 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)

4. Amino Acids: Building Blocks of Protein
Proteins are linear heteropolymers of -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

5. Amino Acids Share Many Features, Differing Only at the R Substituent
FIGURE 3-2 General structure of an amino acid. This structure is common to all but one of the α-amino acids. (Proline, a cyclic amino acid, is the exception.) The R group, or side-chain (red), attached to the α carbon (blue) is different in each amino acid.

6. Amino Acids Have Three Common Functional Groups Attached to the α Carbon
The α carbon always has four substituents and is tetrahedral.
All (except proline) have:
an acidic carboxyl group connected to the α carbon
a basic amino group connected to the α carbon
an α hydrogen connected to the α carbon
The fourth substituent (R) is unique in glycine, the simplest amino acid. The fourth substituent is also hydrogen.

7. All Amino Acids Are Chiral (Except Glycine) Proteins only contain l amino acids
FIGURE 3-3 Stereoisomerism in α-amino acids. (a) The two stereoisomers of alanine, l- and d-alanine, are nonsuperposable mirror images of each other (enantiomers). (b, c) Two different conventions for showing the configurations in space of stereoisomers. In perspective formulas (b), the solid wedge-shaped bonds project out of the plane of the paper, the dashed bonds behind it. In projection formulas (c), the horizontal bonds are assumed to project out of the plane of the paper, the vertical bonds behind. However, projection formulas are often used casually and are not always intended to portray a specific stereochemical configuration.

8. 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)

9. FIGURE 3-5 (part 1) The 20 common amino acids of proteins
FIGURE 3-5 (part 1) The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in pink are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3-1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. The protonated form of histidine is shown above the graph in Figure 3-12b.

10. These amino acid side chains absorb UV light at 270–280 nm
FIGURE 3-5 (part 2) The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in pink are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3-1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. The protonated form of histidine is shown above the graph in Figure 3-12b.
These amino acid side chains absorb UV light at 270–280 nm

11. These amino acids side chains can form hydrogen bonds.
FIGURE 3-5 (part 3) The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in pink are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3-1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. The protonated form of histidine is shown above the graph in Figure 3-12b.
These amino acids side chains can form hydrogen bonds.
Cysteine can form disulfide bonds.

12. FIGURE 3-5 (part 4) The 20 common amino acids of proteins
FIGURE 3-5 (part 4) The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in pink are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3-1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. The protonated form of histidine is shown above the graph in Figure 3-12b.

13. FIGURE 3-5 (part 5) The 20 common amino acids of proteins
FIGURE 3-5 (part 5) The 20 common amino acids of proteins. The structural formulas show the state of ionization that would predominate at pH 7.0. The unshaded portions are those common to all the amino acids; the portions shaded in pink are the R groups. Although the R group of histidine is shown uncharged, its pKa (see Table 3-1) is such that a small but significant fraction of these groups are positively charged at pH 7.0. The protonated form of histidine is shown above the graph in Figure 3-12b.

14. Ionization of Amino Acids
Amino acids contain at least two ionizable protons, each with its own pKa.
The carboxylic acid has an acidic pKa and will be protonated at an acidic (low) pH:
−COOH ↔ COO− + H+
The amino group has a basic pKa and will be protonated until basic pH (high) is achieved:
−NH4+ ↔ NH3 + H+

15. Chemical Environment Affects pKa Values
-carboxy group is much more acidic than in carboxylic acids.
-amino group is slightly less basic than in amines.
FIGURE 3-11 Effect of the chemical environment on pKa. The pKa values for the ionizable groups in glycine are lower than those for simple, methyl-substituted amino and carboxyl groups. These downward perturbations of pKa are due to intramolecular interactions. Similar effects can be caused by chemical groups that happen to be positioned nearby—for example, in the active site of an enzyme.

16. Ionization of Amino Acids
At low pH, the amino acid exists in a positively charged form (cation).
At high pH, the amion acid exists in a negatively charged form (anion).
Between the pKa for each group, the amino acid exists in a zwitterion form, in which a single molecule has both a positive and negative charge.

17. Cation  Zwitterion  Anion
FIGURE 3-10 Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25°C. The ionic species predominating at key points in the titration are shown above the graph. The shaded boxes, centered at about pK1 = 2.34 and pK2 = 9.60, indicate the regions of greatest buffering power. Note that 1 equivalent of OH– = 0.1 M NaOH added.

18. Amino Acids Carry a Net Charge of Zero at a Specific pH (the pI)
Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups.
For amino acids 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.

19. Amino Acids Can Act as Buffers
Amino acids with uncharged side chains, such as glycine, have two pKa values:
The pKa of the -carboxyl group is 2.34.
The pKa of the -amino group is 9.6.
As buffers prevent change in pH close to the pKa, glycine can act as a buffer in two pH ranges.

20. Buffer
Regions
FIGURE 3-10 Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25°C. The ionic species predominating at key points in the titration are shown above the graph. The shaded boxes, centered at about pK1 = 2.34 and pK2 = 9.60, indicate the regions of greatest buffering power. Note that 1 equivalent of OH– = 0.1 M NaOH added.

21. Ionizable Side Chains Also Have pKa and Act as Buffers
Ionizable side chains influence the pI of the amino acid.
Ionizable side chains can be also titrated.
Titration curves are now more complex, as each pKa has a buffering zone of 2 pH units.

22. FIGURE 3-12b Titration curves for (a) glutamate and (b) histidine
FIGURE 3-12b Titration curves for (a) glutamate and (b) histidine. The pKa of the R group is designated here as pKR.

23. How to Calculate the pI When the Side Chain Is Ionizable
Identify species that carries a net zero charge.
Identify the pKa value that defines the acid strength of this zwitterion: (pKR).
Identify the pKa value that defines the base strength of this zwitterion: (pK2).
Take the average of these two pKa values.
What is the pI of histidine?

24. Amino Acids Polymerize to Form Peptides
Peptides are small condensation products of amino acids.
They are “small” compared with proteins (Mw < 10 kDa).
FIGURE 3-13 Formation of a peptide bond by condensation. The α-amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. At physiological pH, the reaction shown here does not occur to any appreciable extent.

25. Peptide Ends Are Not the Same
Numbering (and naming) starts from the amino terminus (N-terminal).
AA AA AA AA AA5
FIGURE 3-14 The pentapeptide serylglycyltyrosylalanylleucine, Ser–Gly–Tyr–Ala–Leu, or SGYAL. Peptides are named beginning with the amino-terminal residue, which by convention is placed at the left. The peptide bonds are shaded in yellow; the R groups are in red.

26. Naming Peptides: Start at the N-terminal
Using full amino acid names:
serylglycyltyrosylalanylleucine
Using the three-letter code abbreviation:
Ser-Gly-Tyr-Ala-Leu
For longer peptides (like proteins) the one- letter code can be used:
SGYAL

27. Peptides: A Variety of Functions
Hormones and pheromones
insulin (think sugar metabolism)
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)

28. Proteins are comprised of:
Polypeptides (covalently linked -amino acids) + possibly:
cofactors
functional non-amino acid component
metal ions or organic molecules
coenzymes
organic cofactors
NAD+ in lactate dehydrogenase
prosthetic groups
covalently attached cofactors
heme in myoglobin
other modifications (posttranslational modifications)

29. Uncommon Amino Acids in Proteins
Not incorporated by ribosomes
except for selenocysteine
Arise by posttranslational modifications of proteins
Reversible modifications, especially phosphorylation, are important in regulation and signaling.

30. Modified Amino Acids Found in Proteins
FIGURE 3-8a Uncommon amino acids. (a) Some uncommon amino acids found in proteins. All are derived from common amino acids. Extra functional groups added by modification reactions are shown in red. Desmosine is formed from four Lys residues (the carbon backbones are shaded in light red). Note the use of either numbers or Greek letters in the names of these structures to identify the altered carbon atoms.

31. Reversible Modifications of Amino Acids
FIGURE 3-8b Uncommon amino acids. (b) Reversible amino acid modifications involved in regulation of protein activity. Phosphorylation is the most common type of regulatory modification.

32. Polypeptide Size and Number Varies Greatly in Proteins
TABLE 3-2
Molecular Data on Some Proteins
Protein
Molecular weight
Number of residues
Number of polypeptide chains
Cytochrome c (human)
12,400
104 1
Ribonuclease A (bovine pancreas)
13,700
124
Lysozyme (chicken egg white)
14,300
129
Myglobin (equine heart)
16,700
153
Chymotrypsin (bovine pancreas)
25,700
245
Chymotrypsinogen (bovine)
Hemoglobin (human)
64,500
574 4
Serum albumin (human)
66,000
609
Hexokinase (yeast)
107,900
972 2
RNA polymerase (E. coli)
450,000
4,158 5
Apolipoprotein B (human)
513,000
4,536
Glutamine synthetase (E. coli)
619,000
5,628 12
Titin (human)
2,993,000
26,926

33. Conjugated Proteins Are Covalently Bound to a Nonprotein Entity
TABLE 3-4
Conjugated Proteins
Class
Prosthetic group
Example
Lipoproteins
Lipids
β1-Lipoprotein of blood
Glycoproteins
Carbohydrates
Immunoglobulin G
Phosphoproteins
Phosphate groups
Casein of milk
Hemoproteins
Heme (iron porphyrin)
Hemoglobin
Flavoproteins
Flavin nucleotides
Succinate dehydrogenase
Metalloproteins
Iron
Zinc
Calcium
Molybdenum
Copper
Ferritin
Alcohol dehydrogenase
Calmodulin
Dinitrogenase
Plastocyanin

34. Common Questions About Peptides and Proteins
What is its sequence and composition?
What is its three-dimensional structure?
How does it find its native fold?
How does it achieve its biochemical role?
How is its function regulated?
How does it interact with other macromolecules?
How is it related to other proteins?
Where is it localized within the cell?
What are its physico-chemical properties?

35. Studying Proteins and Peptides Sometimes Requires Purification from a Mixture
Polypeptides contain differing amino acid sequences.
The sequence and arrangement of amino acids gives the polypeptide a chemical character (i.e., charged, polar, hydrophobic, etc.).
Some polypeptides bind specific targets, which can be used to “fish them out” of a complex mixture.

36. A Mixture of Proteins Can Be Separated
Separation relies on differences in physical and chemical properties:
charge
size
affinity for a ligand
solubility
hydrophobicity
thermal stability
Chromatography is commonly used for preparative separation in which the protein is often able to remain fully folded.

37. Column Chromatography
Column chromatography allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase to mobilize the proteins.
Proteins with a lower affinity for the solid phase will wash off first; proteins with higher affinity will retain on the column longer and wash off later.
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.

38. Separation by Charge: Ion Exchange
FIGURE 3-17a Three chromatographic methods used in protein purification. (a) Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH.

39. Separation by Size: Size Exclusion
FIGURE 3-17b Three chromatographic methods used in protein purification. (b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size.

40. Separation by Binding: Affinity
FIGURE 3-17c Three chromatographic methods used in protein purification. (c) Affinity chromatography separates proteins by their binding specificities. Further details of these methods are given in the text.

41. Electrophoresis for Protein Analysis
Separation in analytical scale is commonly done by electrophoresis.
The electric field pulls proteins according to their charge.
The gel matrix hinders mobility of proteins according to their size and shape.
The gel is commonly polyacrylamide, so separation of proteins via electrophoresis is often called polyacrylamide gel electrophoresis, or PAGE.

42. FIGURE 3-18a Electrophoresis
FIGURE 3-18a Electrophoresis. (a) Different samples are loaded in wells or depressions at the top of the polyacrylamide gel. The proteins move into the gel when an electric field is applied. The gel minimizes convection currents caused by small temperature gradients, as well as protein movements other than those induced by the electric field.

43. SDS PAGE Separates Proteins by Molecular Weight
SDS – sodium dodecyl sulfate – a detergent
SDS micelles bind to proteins and facilitate unfolding.
SDS gives all proteins a uniformly negative charge.
The native shape of proteins does not matter.
The rate of movement will only depend on size: small proteins will move faster.
In this case. the effect of charge is eliminated by binding a negatively charged detergent, SDS, to all the proteins that 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 because these will be forced apart by the SDS.

44. FIGURE 3-18b Electrophoresis
FIGURE 3-18b Electrophoresis. (b) Proteins can be visualized after electrophoresis by treating the gel with a stain such as Coomassie blue, which binds to the proteins but not to the gel itself. Each band on the gel represents a different protein (or protein subunit); smaller proteins move through the gel more rapidly than larger proteins and therefore are found nearer the bottom of the gel.This gel illustrates purification of the RecA protein of Escherichia coli (described in Chapter 25). The gene for the RecA protein was cloned (Chapter 9) so that its expression (synthesis of the protein) could be controlled. The first lane shows a set of standard proteins (of known Mr), serving as molecular weight markers. The next two lanes show proteins from E. coli cells before and after synthesis of RecA protein was induced. The fourth lane shows the proteins in a crude cellular extract. Subsequent lanes (left to right) show the proteins present after successive purification steps. The purified protein is a single polypeptide chain (Mr ~38,000), as seen in the rightmost lane.

45. SDS-PAGE Can Be Used to Calculate the Molecular Weight of a Protein
FIGURE 3–19 Estimating the molecular weight of a protein. The electrophoretic mobility of a protein on an SDS polyacrylamide gel is related to its molecular weight, Mr. (a) Standard proteins of known molecular weight are subjected to electrophoresis (lane 1). These marker proteins can be used to estimate the molecular weight of an unknown protein (lane 2). (b) A plot of log Mr of the marker proteins versus relative migration during electrophoresis is linear, which allows the molecular weight of the unknown protein to be read from the graph. (In similar fashion, a set of standard proteins with reproducible retention times on a size-exclusion column can be used to create a standard curve of retention time versus log Mr. The retention time of an unknown substance on the column can be compared with this standard curve to obtain an approximate Mr.)

46. Isoelectric Focusing Can Be Used to Determine the pI of a Protein
FIGURE 3–20 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A protein mixture is placed on a gel strip containing an immobilized pH gradient. With an applied electric field, proteins enter the gel and migrate until each reaches a pH equivalent to its pI. Remember that when pH = pI, the net charge of a protein is zero.

47. Isoelectric Focusing and SDS-PAGE Are Combined in 2D Electrophoresis
FIGURE 3–21 Two-dimensional electrophoresis. Proteins are first separated by isoelectric focusing in a thin strip gel. The gel is then laid horizontally on a second, slab-shaped gel, and the proteins are separated by SDS polyacrylamide gel electrophoresis. Horizontal separation reflects differences in pI; vertical separation reflects differences in molecular weight. The original protein complement is thus spread in two dimensions. Thousands of cellular proteins can be resolved using this technique. Individual protein spots can be cut out of the gel and identified by mass spectrometry (see Figs 3–30 and 3–31).

48. Aromatic Amino Acids Absorb Light in a Concentration-Dependent Manner
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.
For proteins and peptides with known extinction coefficients (or sequences), concentration can be determined by UV-visible spectrophotometry using the Lambert-Beer law: A = cl

49. FIGURE 3-6 Absorption of ultraviolet light by aromatic amino acids
FIGURE 3-6 Absorption of ultraviolet light by aromatic amino acids. Comparison of the light absorption spectra of the aromatic amino acids tryptophan and tyrosine at pH 6.0. The amino acids are present in equimolar amounts (10–3 M) under identical conditions. The measured absorbance of tryptophan is as much as four times that of tyrosine. Note that the maximum light absorption for both tryptophan and tyrosine occurs near a wavelength of 280 nm. Light absorption by the third aromatic amino acid, phenylalanine (not shown), generally contributes little to the spectroscopic properties of proteins.

50. Specific Activity Describes the Purity of the Protein of Interest
Proteins in a complex mixture often require more than one purification to completely isolate the protein of interest.
During purification, determination of the location of the protein of interest can be performed by tracking its behavior.
If a protein has a specific function (e.g., binding insulin), the fraction that binds insulin best after each purification step will contain the most of the protein of interest.
The function of the protein is called the “activity.”
The ratio of activity to total protein concentration is called the “specific activity.”

51. Specific Activity Describes the Purity of the Protein of Interest
FIGURE 3–22 Activity versus specific activity. The difference between these terms can be illustrated by considering two flasks containing marbles. The flasks contain the same number of red marbles, but different numbers of marbles of other colors. If the marbles represent proteins, both flasks contain the same activity of the protein represented by the red marbles. The second flask, however, has the higher specific activity because red marbles represent a higher fraction of the total.

52. Protein Sequencing
It is essential to further biochemical analysis that we know the sequence of the protein we are studying.
The actual sequence is generally determined from the DNA sequence.
Edman degradation (classical method):
successive rounds of N-terminal modification, cleavage, and identification
can be used to identify protein with known sequence
Mass spectrometry (modern method):
MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence
can be used to determine posttranslational modifications

53. Edman’s Degradation for Protein Sequencing
FIGURE 3–27 The protein sequencing chemistry devised by Pehr Edman. The peptide bond nearest to the amino terminus of the protein or polypeptide is cleaved in two steps. The two steps are carried out under very different reaction conditions (basic conditions in step 1, acidic in step 2), allowing one step to proceed to completion before the second is initiated.

54. MALDI-MS for Protein Sequencing
FIGURE 3–30 Electrospray ionization mass spectrometry of a protein. (a) A protein solution is dispersed into highly charged droplets by passage through a needle under the influence of a high-voltage electric field. The droplets evaporate, and the ions (with added protons in this case) enter the mass spectrometer for m/z measurement. The spectrum generated (b) is a family of peaks, with each successive peak (from right to left) corresponding to a charged species increased by 1 in both mass and charge. The inset shows a computer-generated transformation of this spectrum.
FIGURE 3–31 Obtaining protein sequence information with tandem MS. (a) After proteolytic hydrolysis, a protein solution is injected into a mass spectrometer (MS-1). The different peptides are sorted so that only one type is selected for further analysis. The selected peptide is further fragmented in a chamber between the two mass spectrometers, and m/z for each fragment is measured in the second mass spectrometer (MS-2). Many of the ions generated during this second fragmentation result from breakage of the peptide bond, as shown. These are called b-type or y-type ions, depending on whether the charge is retained on the amino- or carboxyl-terminal side, respectively. (b) A typical spectrum with peaks representing the peptide fragments generated from a sample of one small peptide (21 residues). The labeled peaks are y-type ions derived from amino acid residues. The number in parentheses over each peak is the molecular weight of the amino acid ion. The successive peaks differ by the mass of a particular amino acid in the original peptide. The deduced sequence is shown at the top.

55. Protein Sequences as Clues to Evolutionary Relationships
Sequences of proteins with identical functions from a wide range of species can be aligned and analyzed for differences.
Differences indicate evolutionary divergences.
Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth.

56. Chapter 3: Summary In this chapter, we learned about the:
many biological functions of peptides and proteins
structures and names of amino acids found in proteins
ionization properties of amino acids and peptides
methods for separation and analysis of proteins