In Figure 4.3, the 20 amino acids used for protein synthesis are grouped into dif- ferent classifi cations according to the polarity and structural features of the side chains. These groupings can be helpful in describing common functional roles or metabolic pathways of the amino acids. However, some amino acid side chains fi t into a number of different classifi cations and are therefore grouped differently in dif- ferent textbooks. Two of the characteristics of the side chain that are useful for clas- sifi cation are its pKa and its hydropathic index, which are indicated in Table A4.1, found in the online supplement . The hydropathic index is a scale used to denote
A
B
␣-Carbon Carboxyl
group Amino
group
Side chain H3N
COO– C R
H
+
H3N COOH C R
H
+
H+ pK ˜ 2
H3N COO– C R
H
+
H2N COO– C R
H pK ˜ 9–10 H+
FIG. 4.1. Amino acid structure. A. General structure of the amino acids found in pro- teins. The carbon contains four substituents;
an amino group, a carboxyl group, a hydrogen atom, and a side chain (R). Both the amino and carboxyl groups carry a charge at physi- ological pH. B. Dissociation of the α-carboxyl and α-amino groups of amino acids. At physi- ological pH (⬃7), a form in which both the α-carboxyl and α-amino groups are charged predominates. Some amino acids also have ionizable groups on their side chains.
H2O O–
R1 C C H3N
H O
+
H C C H3N
R2 O O–
+
R1 C C H3N
H
H O R2
N H C C O–
O
+
+
FIG. 4.2. Peptide bonds. Amino acids in a polypeptide chain are joined through peptide bonds between the carboxyl group of one amino acid and the amino group of the next amino acid in the sequence.
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N o n p o l a r, A l i p h a t i c
C H COO–
H
C H COO–
CH3
Cyclic
H3N+ H3N+ C H
COO– CH2 CH2 H2N
H2C
+
Valine (val, V) C H COO–
CH CH3 CH3 H3N+
Leucine (leu, L)
Isoleucine (ile, l)
C H COO–
C CH2 CH3 C H
COO–
CH2 CH H3N+
CH3 CH3
H3N+
CH3 H
Alanine (ala, A) Glycine
(gly, G)
Branched-chain
Proline (pro, P)
Tyrosine (tyr, Y)
Nonpolar More Polar
A ro m a t i c
Tryptophan (trp, W) Phenylalanine
(phe, F)
C H COO–
CH2
H3N+ C H
COO–
CH2 H3N+ C H
COO–
CH2 H3N+
OH
NH C CH
C h a r g e d
Aspartate (asp, D)
Negative (Acidic) Positive (Basic)
Glutamate (glu, E)
Arginine (arg, R)
Lysine (lys, K)
Histidine (his, H) C
COO–
CH2 COO–
H
H3N+ C
COO–
CH2 CH2 COO–
H
H3N+ C
COO–
CH2 CH2 CH2 NH C NH2 NH2
H H3N+
+
C COO–
CH2 CH2 CH2 CH2 NH3 H H3N+
+
C COO–
CH2 H H3N+
C
HC CH NH
N C H COO–
CH2 CH2 S CH3 Methionine
(met, M)
H3N+ C H
COO–
CH2 SH
Cysteine (cys, C) H3N+
S u l f u r- C o n t a i n i n g P o l a r, U n c h a r g e d
Serine (ser, S)
Threonine (thr, T) Asparagine
(asn, N)
Glutamine (gln, Q)
C H COO–
CH2 CH2 C C H
COO–
CH2 C H3N+
H2N O
H2N O
H3N+ C H
COO–
CH2OH
H3N+ C H
COO–
C CH3 H3N+
OH H
FIG. 4.3. The side chains of the amino acids. The side chains are highlighted. The amino acids are grouped by the polarity and structural features of their side chains. These groupings are not absolute, however. Tyrosine and tryptophan, often listed with the nonpolar amino acids, are more polar than other aromatic amino acids because of their phenolic and indole rings, respectively. The single- and three-letter codes are also indicated for each amino acid.
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CHAPTER 4 ■ AMINO ACIDS AND PROTEINS 49
the hydrophobicity of the side chain; the more positive the hydropathic index, the greater the tendency to cluster with other nonpolar molecules and exclude water in the hydrophobic effect. These hydrophobic side chains tend to occur in membranes or in the center of a folded protein, where water is excluded. The more negative the hydropathic index of an amino acid, the more hydrophilic is its side chain.
The names of the different amino acids have been given three-letter and one- letter abbreviations (see Fig. 4.3). The three-letter abbreviations use the fi rst two letters in the name plus the third letter of the name or the letter of a characteristic sound, such as “trp” for tryptophan. The one-letter abbreviations use the fi rst letter of the name of the most frequent amino acid in proteins (such as an “A” for alanine).
If the fi rst letter has already been assigned, the letter of a characteristic sound is used (such as an “R” for arginine). Single-letter abbreviations are usually used to denote the amino acids in a polypeptide sequence.
A. Nonpolar, Aliphatic Amino Acids
Glycine is the simplest amino acid and really does not fi t well into any classifi cation because its side chain is only a hydrogen atom. Because the side chain of glycine is so small compared to that of other amino acids, it causes the least amount of steric hindrance in a protein (i.e., it does not signifi cantly impinge on the space occupied by other atoms or chemical groups). Therefore, glycine is often found in bends or in the tightly-packed chains of fi brous proteins.
Alanine and the branched chain amino acids (valine, leucine, and isoleucine) have bulky, nonpolar, aliphatic (open-chain hydrocarbon) side chains and exhibit a high degree of hydrophobicity. Electrons are shared equally between the carbon and hydrogen atoms in these side chains, so that they cannot hydrogen bond with water, and therefore, the side chains do not interact with water. Within proteins, these amino acid side chains will cluster together to form hydrophobic cores. Their association is also promoted by van der Waals forces between the positively charged nucleus of one atom and the electron cloud of another. This force is effective over short distances when many atoms pack closely together.
The role of proline in amino acid structure differs from those of the nonpolar amino acids. The amino acid proline contains a ring involving its α-carbon and its α-amino group, which are part of the peptide backbone. It is an imino acid. This rigid ring causes a kink in the peptide backbone that prevents it from forming its usual confi guration, and it will restrict the conformation of the protein at that point.
B. Aromatic Amino Acids
The aromatic amino acids have been grouped together because they all contain ring structures with similar properties, but their polarity differs a great deal. The aromatic ring is a six-member carbon-hydrogen ring with three conjugated double bonds (the benzene ring or phenyl group). These hydrogen atoms do not participate in hydro- gen bonding. The substituents on this ring determine whether the amino acid side chain engages in polar or hydrophobic interactions. In the amino acid phenylalanine, the ring contains no substituents, and the electrons are shared equally between the carbons in the ring, resulting in a very nonpolar hydrophobic structure in which the rings can stack on each other (Fig. 4.4A). In tyrosine, a hydroxyl group on the phenyl ring engages in hydrogen bonds, and the side chain is therefore more polar and more hydrophilic. The more complex ring structure in tryptophan is an indole ring with a nitrogen that can engage in hydrogen bonds. Tryptophan is therefore also more polar than phenylalanine.
C. Aliphatic, Polar, Uncharged Amino Acids
Amino acids with side chains that contain an amide group (asparagine and glu- tamine) or a hydroxyl group (serine and threonine) can be classifi ed as aliphatic, polar, uncharged amino acids. Asparagine and glutamine are amides of the amino acids aspartate and glutamate. The hydroxyl groups and the amide groups in the side
A. Hydrophobic interaction
B. Hydrogen bonds Phenylalanine
side chains Peptide
backbone
Peptide backbone
Side chains
CH2 CH2
O
C O R
N O
H R H H
FIG. 4.4. Hydrophobic and hydrogen bonds.
A. Strong hydrophobic interactions occur with the stacking of aromatic groups in phenyl- alanine side chains. B. Examples of hydrogen bonds in which a hydrogen atom is shared by a nitrogen in the peptide backbone and an oxy- gen atom in an amino acid side chain or be- tween an oxygen in the peptide backbone and an oxygen in an amino acid side chain.
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chains allow these amino acids to form hydrogen bonds with water, with each other and the peptide backbone, or with other polar compounds in the binding sites of the proteins (see Fig. 4.4B). As a consequence of their hydrophilicity, these amino acids are frequently found on the surface of water-soluble globular proteins.
D. Sulfur-containing Amino Acids
Both cysteine and methionine contain sulfur. The side chain of cysteine contains a sulfhydryl group that has a pKa of about 8.4 for dissociation of its hydrogen, so cyste- ine is predominantly undissociated and uncharged at the physiological pH of 7.4. The free cysteine molecule in solution can form a covalent disulfi de bond with another cysteine molecule through spontaneous (nonenzymatic) oxidation of their sulfhydryl groups. The resultant amino acid, cystine, is present in blood and tissues, and is not very water-soluble. In proteins, the formation of a cystine disulfi de bond between two appropriately positioned cysteine sulfhydryl groups often plays an important role in holding two polypeptide chains or two different regions of a chain together (Fig. 4.5).
Methionine, although it contains a sulfur group, is a nonpolar amino acid with a large bulky side chain that is hydrophobic. It does not contain a sulfhydryl group, and can- not form disulfi de bonds. Its important and central role in metabolism is related to its ability to transfer the methyl group attached to the sulfur atom to other compounds.
E. The Acidic and Basic Amino Acids
The amino acids aspartate and glutamate have carboxylic acid groups that carry a negative charge at physiological pH (see Fig. 4.3). The basic amino acids histidine, lysine, and arginine have side chains containing nitrogen that can be protonated and positively charged at physiological and lower pH values.
The positive charges on the basic amino acids enables them to form ionic bonds (electrostatic bonds) with negatively charged groups, such as the side chains of acidic amino acids or the phosphate groups of coenzymes (Fig. 4.6). The acidic and basic amino acid side chains also participate in hydrogen bonding and the forma- tion of salt bridges (such as the binding of an inorganic ion like Na⫹ between two partially or fully negatively charged groups).
The charge on these amino acids at physiological pH is a function of their pKas for dissociation of protons from the α-carboxylic acid groups, the α-amino groups, and the side chains. The titration curve of histidine illustrates the changes in amino acid structure that occur as the pH of the solution is changed from less than 1 to 14 by the addition of hydroxide ions (Fig. 4.7). At low pH, all groups carry protons, amino groups have a positive charge, and carboxylic acid groups have zero charge.
As the pH is increased by the addition of alkali (OH⫺), the proton dissociates from the carboxylic acid group, and its charge changes from zero to negative, with a pKa
of about 2, the pH at which 50% of the protons have dissociated.
The histidine side chain is an imidazole ring with a pKa of about 6 that changes from a predominantly protonated positively charged ring to an uncharged ring at this pH. The amino group on the α-carbon titrates at a much higher pH (between 9 and 10), and the charge changes from positive to zero as the pH rises. The pH at which the net charge on the molecules in solution is zero is called the isoelectric point (pI).
Sulfhydryl groups
Disulfide H3N
SH CH CH2
Cysteine Cysteine
COO–
+
H3N SH
CH CH2
COO–
+
Oxidation Reduction
H3N CH2 CH COO–
+
S
Cystine H3N
S
CH CH2
COO–
+
FIG. 4.5. A disulfi de bond. Covalent disulfi de bonds may be formed between two molecules of cysteine or between two cysteine residues in a protein. The disulfi de compound is called cystine. The hydrogens of the cysteine sulfhy- dryl groups are removed during oxidation.
CH2
O– C CH2 CH2 CH2
CH2 NH3
O
+
FIG. 4.6. Electrostatic interaction between the positively charged side chain of lysine and the negatively charged side chain of aspartate.
Will S. has sickle cell anemia caused by a point mutation in his DNA that changes the sixth amino acid in the β-globin chain of hemoglobin from glutamate to valine. What difference would you expect to fi nd in the noncovalent bonds formed by these two amino acids?
Electrophoresis, a technique used to separate proteins on the basis of charge, has been extremely useful in medicine to identify proteins with different amino acid composition. The net charge on a protein at a certain pH is a summation of all of the positive and negative charges on all of the ionizable amino acid side chains plus the N-terminal amino and C-terminal carboxyl groups. Theoretically, the net charge of a protein at any pH could be determined from its amino acid composition by calculating the concentration of positively and negatively charged groups from the Henderson-Hasselbalch equation (see Chapter 2). However, hydrogen bonds and ionic bonds between amino acid side chains in the protein make this calculation unrealistic.
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CHAPTER 4 ■ AMINO ACIDS AND PROTEINS 51
At this pH, the molecules will not migrate in an electric fi eld toward either a positive pole (cathode) or a negative pole (anode) because the number of negative charges on each molecule is equal to the number of positive charges.
Amino acid side chains change from uncharged to negatively charged or from positively charged to uncharged as they release protons. The acidic amino acids lose a proton from their carboxylic acid side chains at a pH of about 4 and are thus negatively charged at pH 7.4. Cysteine and tyrosine lose protons at their pKa (⬃8.4 and 10.5, respectively), so their side chains are uncharged at physiological pH. His- tidine, lysine, and arginine side chains change from positively charged to neutral at their pKa. The side chains of the two basic amino acids arginine and lysine have pKa values above 10, so that the positively charged form always predominates at physiological pH. The side chain of histidine (pKa ⬃6.0) dissociates near physi- ological pH, so only a portion of the histidine side chains carry a positive charge (see Fig. 4.7).
In proteins, only the amino acid side chains and the amino group at the amino terminal and carboxyl group at the carboxyl terminal have dissociable protons. All of the other carboxylic acid and amino groups on the α-carbons are joined in peptide bonds that have no dissociable protons. The amino acid side chains might have a very different pKa than those of the free amino acids if they are involved in hydrogen or ionic bonds with other amino acid side chains. The pKa of the imidazole group of histidine, for example, is often shifted to a higher value (between 6 and 7) so that it adds and releases a proton in the physiological pH range.
III. VARIATIONS IN PRIMARY STRUCTURE
Although almost every amino acid in the primary structure of a protein contributes to its conformation (three-dimensional structure), the primary structure of a protein
Glutamate carries a negative charge on its side chain at physiological pH and, thus, can engage in ionic bonds or hydrogen bonds with water or other side chains. Valine is a hydrophobic amino acid and, therefore, tends to interact with other hy- drophobic side chains to exclude water. (The effect of this substitution on hemoglobin struc- ture is described in more detail in Chapter 7.)
+
N HN pKa1 (␣COOH) = 1.8
pKa3 (␣NH+ 3) = 9.3
pKa2 (R group) = 6.0 pI
Equivalents of OH–
0 0.5 1.0 1.5 2.0 2.5 3.0
pH
2 4 6 8 10 12 14
H3N CH COOH
+
Below pH 1.8
NH+ HN
CH2
Predominant species H3N CH
COO–
+
Between pH 1.8 and 6.0
H3N CH COO–
Between pH 6.0 and 9.3 NH+
HN
N HN
H2N CH COO–
CH2 CH2 CH2
Above pH 9.3 pKa2
pKa1 pKa3
FIG. 4.7. Titration curve of histidine. The ionic species that predominates in each region is shown below the graph. pI is the isoelectric point (the pH at which there is no net charge on the molecule).
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can vary to some degree between species. Even within the human species, the amino acid sequence of a normal functional protein can vary somewhat among individuals, tissues of the same individual, and the stage of development. These variations in the primary structure of a functional protein are tolerated if they are confi ned to noncrit- ical regions (called variant regions), if they are conservative substitutions (replace one amino acid with one of similar structure), or if they confer an advantage. If many different amino acid residues are tolerated at a position, the region is called hypervariable. In contrast, the regions that form binding sites or are critical for forming a functional three-dimensional structure are usually invariant regions that have exactly the same amino acid sequence from individual to individual, tissue to tissue, or species to species.
A. Polymorphism in Protein Structure
Within the human population, the primary structure of a protein may vary slightly among individuals. The variations generally arise from mutations in DNA that are passed to the next generation. The mutations can result from the substitution of one base for another in the DNA sequence of nucleotides (a point mutation), from deletion or insertions of bases into DNA, or from larger changes (see Chapter 12).
For many alleles, the variation has a distinct phenotypic consequence that contrib- utes to our individual characteristics, produces an obvious dysfunction (a congen- ital or genetically inherited disease), or increases susceptibility to certain diseases.
A defective protein may differ from the most common allele by as little as a single amino acid that is a nonconservative substitution (replacement of one amino acid with another of a different polarity or very different size) in an invariant region. Such mutations might affect the ability of the protein to carry out its function, catalyze a particular reaction, reach the appropriate site in a cell, or be degraded. For other proteins, the variations appear to have no signifi cance.
Variants of an allele that occur with a signifi cant frequency in the population are referred to as polymorphisms. Thus far in studies of the human genome, almost one-third of the genetic loci appear to be polymorphic. When a particular variation of an allele, or polymorphism, increases in the general population to a frequency of more than 1%, it is considered stable. The sickle cell allele is an example of a point mutation that is stable in the human population. Its persistence is probably due to selective pressure for the heterozygous mutant phenotype, which confers some pro- tection against malaria.
For the most part, human chromo- somes occur as homologous pairs, with each member of a pair contain- ing the same genetic information. One member of the pair is inherited from the mother and one from the father. Genes are arranged linearly along each chromosome. A genetic locus is a specifi c position or location on a chromosome.
Alleles are alternative versions of a gene at a given locus. For each locus (site), we have two alleles of each gene, one from our mother and one from our father. If both alleles of a gene are identical, the individual is homozygous for this gene; if the alleles are different, the indi- vidual is heterozygous for this gene. Will S.
has two identical alleles for the sickle variant of the β-globin gene that results in substitution of a valine for a glutamate residue at the sixth position of the β-globin chain. He is therefore homozygous for the sickle cell allele and has sickle cell anemia. Individuals with one normal gene and one sickle cell allele are heterozy- gous. They are carriers of the disease and have sickle cell trait.
Will S.’s hemoglobin, HbS, is composed of two normal α chains and two β-globin chains with the sickle cell variant (α2β2S). The change in amino acid composition from a glutamate to a valine in the β chain allows sickle hemoglobin to be sepa- rated from normal adult hemoglobin (HbA, or [α2β2A]) by electrophoresis. In electrophore- sis, an aliquot of blood or other solution containing proteins is applied to a support, such as paper or a gel. An electrical fi eld is applied and the proteins migrate a distance toward the anode (negative pole) or cathode (positive pole) depending on their net charge. Since βS contains one less negative charge than βA, it will migrate differently in an electric fi eld.
Individuals with sickle cell trait are heterozygous and have both HbA and HbS, plus small amounts of fetal hemoglobin, HbF (α2γ2).
In heterozygous individuals with sickle cell trait, the sickle cell allele provides some pro- tection against malaria. In Will S. and other homozygous individuals with sickle cell ane- mia, however, the red blood cells sickle more frequently than in heterozygotes, especially under conditions of low oxygen tension (see Chapter 5). The result is a vaso-occlusive cri- sis in which the sickled cells clog capillaries and prevent oxygen from reaching cells (isch- emia), thereby causing pain. The enhanced destruction of the sickled cells by the spleen results in anemia. Consequently, the sickle cell allele is of little advantage to homozygous individuals.
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