We have seen that one class of biopolymers,
the nucleic acids, stores and transmits the genetic information of the cell. Much of that information is expressed in another class of biopolymers, the proteins. Proteins play an enor-mous variety of roles: Some carry out the transport and storage of small mole-cules; others make up a large part of the structural framework of cells and tissues.Muscle contraction, the immune response, and blood clotting are all mediated by proteins. An important class of proteins is the enzymes—the catalysts that pro-mote the tremendous variety of reactions that are required to support the living state. Each type of cell in every organism has several thousand kinds of proteins to serve these many functions.
In keeping with the multiplicity of their functions, proteins are extremely com-plex molecules. This comcom-plexity is illustrated in Figure 5.1, which depicts the molec-ular structure of myoglobin, a relatively small protein that functions primarily in oxygen binding and storage in animal tissues. In this and the following three chapters, we analyze in detail the structures and functions of a handful of proteins, including myoglobin. We will see that although there are general features of protein structure shared by most proteins, each protein has a distinct structure that is optimally suited to its function. Protein structures may appear at first glance to be hopelessly complex;
however, there is an elegant and readily comprehensible logic to protein structure, which we will describe here and in Chapter 6. We begin with a description of the simple “building blocks” that are found in all proteins: the amino acids.
Amino Acids
Structure of the -Amino Acids
All proteins are polymers, and the monomers that combine to make them are -amino acids. A general representation of an -amino acid is shown in Figure 5.2a.
The amino group is attached to the -carbon, the carbon next to the carboxylic acid group; hence the name -amino acid. To the -carbon of every amino acid are alsoa a aa A
A
Introduction to Proteins:
The Primary Level
of Protein Structure
AMINO ACIDS
137
FIGURE 5.1
The three-dimensional structure of the globular protein myoglobin. This molecular model was generated from the X-ray crystal structure determined by H. C. Watson and J. C. Kendrew (PDB ID: 1MBN), and it shows the heavy atoms (i.e., non-hydrogen atoms) in sperm whale myoglobin. The amino acid atoms are shown as sticks, where carbon atoms are gray, oxygen atoms are red, nitrogen atoms are blue, and sulfur atoms are yellow. The heme atoms are shown in a space-filling display that represents the van der Waals surface of each atom. The orange sphere in the center of the heme is the Fe2⫹ion that binds a molecule of oxygen (the oxygen-binding site is obscured by the heme). The orientation of the protein in this figure is the same at that shown in the paint-ing by Irvpaint-ing Geis in Chapter 1 (Figure 1.5).
attached a hydrogen atom and a side chain (“R” group). Different -amino acids are distinguished by their different side chains. We can write the general structure for an
-amino acid as shown in Figure 5.2a. This representation, although chemically cor-rect, ignores the conditions in vivo. As pointed out in Chapter 2, most biochemistry occurs in the physiological pH range near neutrality. The ’s of the carboxylic acid and amino groups of the -amino acids are about 2 and 10, respectively. Therefore, near neutral pH the carboxylic acid group will have lost a proton, and the amino group will have picked up a proton, to yield the zwitterion form shown in Figure 5.2b.
This is the form in which we will customarily write amino acid structures.
Twenty different kinds of amino acids are commonly incorporated into proteins during the process of translation (see Figure 4.23 page 112). The complete structures
a pKa
a
a
138
CHAPTER 5 INTRODUCTION TO PROTEINS: THE PRIMARY LEVEL OF PROTEIN STRUCTUREAbbreviations Occurrencec
1- and 3-letter pKaof pKaof pKaof Ionizing ResiduebMass in Proteins
Name codes -COOH Group -NH3+Group Side Chaina (daltons) (mol %)
Alanine A, Ala 2.3 9.7 — 71.08 8.7
Arginine R, Arg 2.2 9.0 12.5 156.20 5.0
Asparagine N, Asn 2.0 8.8 — 114.11 4.2
Aspartic acid D, Asp 2.1 9.8 3.9 115.09 5.9
Cysteine C, Cys 1.8 10.8 8.3 103.14 1.3
Glutamine Q, Gln 2.2 9.1 — 128.14 3.7
Glutamic acid E, Glu 2.2 9.7 4.2 129.12 6.6
Glycine G, Gly 2.3 9.6 — 57.06 7.9
Histidine H, His 1.8 9.2 6.0 137.15 2.4
Isoleucine I, Ile 2.4 9.7 — 113.17 5.5
Leucine L, Leu 2.4 9.6 — 113.17 8.9
Lysine K, Lys 2.2 9.0 10.0 128.18 5.5
Methionine M, Met 2.3 9.2 — 131.21 2.0
Phenylalanine F, Phe 1.8 9.1 — 147.18 4.0
Proline P, Pro 2.0 10.6 — 97.12 4.7
Serine S, Ser 2.2 9.2 — 87.08 5.8
Threonine T, Thr 2.6 10.4 — 101.11 5.6
Tryptophan W, Trp 2.4 9.4 — 186.21 1.5
Tyrosine Y, Tyr 2.2 9.1 10.1 163.18 3.5
Valine V, Val 2.3 9.6 — 99.14 7.2
aApproximate values found for side chains on the free amino acids.
bTo obtain the mass of the amino acid itself, add the mass of a molecule of water, 18.02 daltons. The values given are for neutral side chains; slightly different values will apply at pH values where protons have been gained or lost from the side chains.
cAverage for a large number of proteins. Individual proteins can show large deviations from these values. Data from Journal of Chemical Information and Modeling 50:690–700, J. M. Otaki, M. Tsutsumi, T. Gotoh, and H. Yamamoto, Secondary structure characterization based on amino acid composition and availability in proteins. © 2010 American Chemical Society.
W. P. Jencks and J. Regenstein (1976) Ionization constants of acids and bases in Handbook of Biochemistry and Molecular Biology, 3rd ed., G. Fasman (ed.), CRC Press, Boca Raton, FL.
a a
Side chain
(a)
(b)
H R
OH α-carbon O
H2N
H R
O O H3N
zwitterion
− +
FIGURE 5.2
The structure of an -amino acid.(a) A general representation of a nonionized -amino acid showing the carboxylic acid group, the -amino group, and a hydrogen bonded to the -carbon, as well as the side chain (R group) that gives the amino acid its unique properties. (b) An amino acid shown as a zwitterion at neutral pH. Under physiological conditions, amino acids exist as zwitterions in which the -carboxylic acid group has lost a proton and the -amino group has gained one. Note that the negative charge on the -carboxylate is delocalized over the two oxygen atoms. The stereo-chemistry shown in this figure is that for the -amino acids found in biosynthetic proteins.
a a a a
a aa a
of these amino acids are shown in Figure 5.3 and other important data are given in Table 5.1. At least two additional amino acids, selenocysteine and pyrrolysine, are encoded genetically and incorporated into proteins; however, they are found in a rel-atively small number of proteins. For the purposes of this introductory discussion we will focus our attention on the twenty common amino acids shown in Figure 5.3.
Stereochemistry of the -Amino Acids
The asymmetry of biomolecules plays a critical role in determining their struc-tures and functions; thus, familiarity with the basic stereochemistry of amino acids is necessary for an understanding of the biochemistry of proteins.
The four groups shown in Figure 5.2a are bonded to the central -carbon in a tetrahedral arrangement, as is predicted for an hybridized carbon atom. In Figure 5.2 the projection of these groups around the –carbon is represented as follows: The lines represent bonds in the plane of the page, the solid wedges rep-resent bonds projecting forward from the page, and the dashed wedges reprep-resent bonds projecting behind the page. When a carbon atom has four different sub-stituents attached to it, it is said to be chiral, or a stereocenter, or, preferably, an asymmetric carbon. In Figure 5.3, the stereochemistry of the amino acids is shown by a convention known as the Fischer projection. In a Fischer projection the bonds are all represented as solid lines, where the horizontal bonds project forward from the page and the vertical bonds project behind the page. To help you visualize the Fischer projection convention, we have drawn in Figure 5.4 the general structure of an amino acid in a ball-and-stick rendering as well as a Fischer projection that includes solid and dashed wedges. Note that the spatial orientation of the four groups bound to the Cais the same in Figures 5.2, 5.3, and 5.4.
(Ca) a
sp3
a
a
TABLE5.1 Properties of the common amino acids found in proteins
AMINO ACIDS
139
C COO−
H
Glycine (Gly) G H
H3N+ C COO−
CH3
Alanine (Ala) A H
H3N+ C COO−
CH CH3 CH3
Valine (Val) V H
H3N+ C COO−
CH2
Leucine (Leu) L CH CH3 CH3
H
H3N+ C COO−
Isoleucine (Ile) I CH CH3
CH2 CH3
H H3N+
ALIPHATIC AMINO ACIDS
C COO−
Serine (Ser) S H
H3N+ C COO−
CH2 OH
Cysteine (Cys) C H
H3N+ C COO−
CH3
Threonine (Thr) T H
H3N+ C COO−
CH2 CH2
Methionine (Met) M CH3
H H3N+
AMINO ACIDS WITH HYDROXYL- OR SULFUR-CONTAINING SIDE CHAINS
C COO−
Phenylalanine (Phe) F H
+
H3N CH2
AROMATIC AMINO ACIDS
CH2 SH
HCOH
S
C COO−
CH2 CH2
CH2
Proline (Pro) P H H2N+
CYCLIC AMINO ACID
C COO−
Histidine (His) H H
+
H3N CH2 BASIC AMINO ACIDS
C COO−
Tyrosine (Tyr) Y H
+
H3N CH2 OH
C COO−
Tryptophan (Trp) W H
+
H3N CH2 H N
N HN
C COO−
CH2 CH2
Lysine (Lys) K CH2 CH2
H
+
H3N NH+ 3
C COO−
CH2 CH2
Arginine (Arg) R CH2
H
+
H3N
C NH+ 2 NH2
NH
ACIDIC AMINO ACIDS AND THEIR AMIDES
C COO−
CH2 CH2
Glutamic acid (Glu) E H
H3N+
O− O
C
C COO−
CH2
Aspartic acid (Asp) D H
H3N+
O− O
C
C COO−
CH2 CH2
Glutamine (Gln) Q H
H3N+
O
C COO−
CH2
Asparagine (Asn) N H
H3N+
O C
NH2 C
NH2
FIGURE 5.3
The 20 common amino acids found in proteins. The 20 common –amino acids that are incorporated into proteins are shown in Fischer projections and arranged here in the order in which they are discussed in the text. The “side chain” or “R-group” of each amino acid is high-lighted in orange. Below each amino acid are its name, its three-letter abbreviation, and its one-letter abbreviation.
a
Proteins are polymers of a-amino acids.
There are 20 common a-amino acids that are the major building blocks of proteins.
All a-amino acids except glycine contain an asymmetric a-carbon, thus both Land D
enantiomers are possible. However, in the vast majority of proteins, only the
L-enantiomers are found.
If a molecule contains one asymmetric carbon, two distinguishable stereoisomers exist; these are nonsuperimposable mirror images of one another, or enantiomers, as shown in Figure 5.5. The stereoisomers of alanine shown in Figure 5.5 are called the LandDenantiomers.* The LandDenantiomers can be distin-guished from one another experimentally because their solutions rotate plane polar-ized light in opposite directions. For this reason, enantiomers are sometimes called optical isomers. All amino acids except glycine can exist in DandLforms because in each case the -carbon is asymmetric. Glycine is the sole exception because two ofa
*Those who are familiar with modern organic chemistry will know that there are two com-monly used systems for distinguishing stereoisomers—the older D–Lsystem and the newer, more comprehensive R–S system (Cahn-Ingold-Prelog system). Both are discussed in more detail in Chapter 9.
the four groups bonded to the -carbon are the same (i.e., there are two H atoms), eliminating the asymmetry.
Chemical analysis of naturally occurring proteins shows that nearly all of the amino acids found in proteins are of the Lform. Random mixtures ofD-and
L-amino acids could not reproducibly form protein structures as well-defined as the structure of myoglobin shown in Figure 5.1. Cell viability depends on protein function, and protein function depends on the ability of a protein to adopt a well-defined active structure; thus, the need for cells to produce many structurally identical copies of a given protein is absolute. For this reason, nature uses only
L-amino acids in protein biosynthesis. How the absolute preference for the L-isomer over the D-isomer evolved is puzzling. Indeed, we shall find that each of the three major classes of biological macromolecules exhibits a strong preference for one stereoisomer class or the other. Most naturally occurring polysaccharides prefer
D-sugars, as do DNA and RNA. It may be that productive interaction between these substances was established early in the evolution of life; but, why was a par-ticular set of enantiomers chosen at all? It is hard to see how L-amino acids have any inherent selective advantage over the D-isomers for biological function.
Indeed,D-amino acids exist in nature, and some play important biochemical roles (some examples are given in Table 5.2), but they are rarely found in proteins.
Many scientists have attempted to provide explanations for this “handedness pref-erence” in biology. Most point to an intrinsic asymmetry in the behavior of subnu-clear particles, a kind of asymmetry that gives electrons emitted in decay a preferen-tial left-hand spin. Such influences are very weak but might, in a competition between primitive organisms using L- or D-proteins, give a slight advantage to one or the other.
After billions of generations, even a small advantage can become overwhelming.
Using peptide synthesis methods described in Tools of Biochemistry 5C, it is possible to chemically synthesize proteins using all D-amino acids. These structures are the mirror images of the corresponding natural proteins. One such D-protein synthesized in the laboratory of Stephen Kent is the mirror image of a protease (a protein-cleaving enzyme) from the human immunodeficiency virus, HIV (see References). Whereas its natural L-counterpart cleaves natural L-proteins, this syn-thetic enzyme will cleave only those containing D-amino acids. The results of this experiment suggest that life would be possible for cells that made proteins from onlyD-amino acids rather than L-amino acids.
b a
140
CHAPTER 5 INTRODUCTION TO PROTEINS: THE PRIMARY LEVEL OF PROTEIN STRUCTUREH H
O O
H +
−
(a)␣-Amino acid N
Cα
R H
C
(b) Representation of an amino acid in Fischer projection
C␣
R H
COO
⫽
NH3
Cα H NH3
COO +
R
+
− −
FIGURE 5.4
Three-dimensional representations of -amino acids.(a) This ball-and-stick model shows the three-dimensional arrangement of the atoms. The -carbon is asymmetric, with tetrahedral bonding. (b) In a Fis-cher projection (left) the horizontal bonds are project-ing toward the viewer and the vertical bonds are pro-jecting away from the viewer. This orientation of bonds in the Fischer projection is represented on the right by solid and dashed wedges.
a a
C
(a)
(b) H
H
H H
H
H H
H H
H H
H H
C H
C
O O O O
N N
C
L-Alanine D-Alanine
L-Alanine D-Alanine
H NH3
COO
H3C CH3
NH3
COO H
+ +
−
− −
−
Cα Cα
Cα + +
Cα FIGURE 5.5
Stereoisomers of -amino acids.(a)L-alanine and its enantiomer D-alanine are shown as ball-and-stick models. The alanine side chain is CH3. The two mod-els are mirror images, which are not superimposable.
The plane of mirror symmetry is represented by the vertical dashed line (red). (b) The same two enan-tiomers in a Fischer projection.
a
Name Formula Biochemical Source, Function
-Alanine Found in the vitamin pantothenic acid and in some important
natural peptides
D-Alanine In polypeptides in some bacterial cell walls
-Aminobutyric Brain, other animal tissues; functions as neurotransmitter
acid
D-Glutamic acid In polypeptides in some bacterial cell walls
L-Homocysteine Many tissues; precursor for methionine biosynthesis
L-Ornithine Many tissues; an intermediate in arginine synthesis
Sarcosine Many tissues; intermediate in amino acid synthesis
L-Thyroxine Thyroid gland; is thyroid hormone (I = iodine)
g b
TABLE5.2 Some biologically important amino acids not typically found in proteins
AMINO ACIDS
141
Reza Ghadiri and coworkers have shown that a homochiral 32-residue peptide (i.e., composed of either all L- or all D-amino acids) preferentially catalyzes replica-tion of homochiral products from a racemic mixture of peptide fragments. These experiments do not explain why L-amino acids are preferred over D-amino acids;
but, they show that once the preference is established, there is a natural tendency to amplify homochiral sequences (in this case the sequences containing only
L-amino acids).
The preference for L-amino acids in natural proteins has two important con-sequences, which we will discuss further in subsequent chapters:
1. The surface of any given protein, which is where the interesting biochem-istry occurs, is asymmetric. This asymmetry is the basis for the highly spe-cific molecular recognition of binding targets by proteins.
2. The stereochemistry of the amino acids plays an important role in the for-mation of so-called “secondary structure” (i.e., helices and strands) and thereby the overall structure of proteins.
b a
H3N+ CH2 CH2 COO−
H C
COO− NH3 CH3
+
H3N+ CH2 CH2 CH2 COO−
H C
COO− NH3 CH2
CH2 COO−
+
H C COO−
CH2 CH2SH H3N+
H C COO−
CH2 H3N
CH2 CH2NH3
+
+
CH3 N COO−
H CH2
H C COO−
CH2 H3N
O OH
+ I
I
I
I
The stereochemistry of the amino acids plays an important role in the formation of the structure of proteins.
Properties of Amino Acid Side Chains:
Classes of -Amino Acids
The 20 common amino acids contain, in their 20 different side chains, a remarkable collection of chemical groups. It is this diversity of the side chains that allows proteins to exhibit such a great variety of structures and properties. If we examine Figure 5.3, it becomes evident that there are several different classes of side chains, distinguished by their dominant chemical features. These features include hydrophobicity or hydrophilicity, polar or nonpolar character, and the presence or absence of ionizable groups. Many ways have been proposed to group the amino acids into classes, but none is wholly satisfactory. We shall discuss the amino acids in the order shown in Figure 5.3, which proceeds from the simplest to the more complex.
Amino Acids with Aliphatic Side Chains
Glycine, alanine, valine, leucine, and isoleucine have aliphatic side chains. As we progress from left to right along the top row of Figure 5.3, the R group becomes more extended and more hydrophobic. Isoleucine, for example, has a much greater tendency to transfer from water to a hydrocarbon solvent than does ala-nine. The more hydrophobic amino acids such as isoleucine are usually found within the core of a protein molecule, where they are shielded from water. Proline, which has a secondary –amino group, is difficult to fit into any category. It is the only amino acid in this group in which the side chain forms a covalent bond with the –amino group. The proline side chain has a primarily aliphatic character;
however, it is frequently found on the surfaces of proteins due to its unique structural constraints. The rigid ring of proline is well-suited to those sites in a protein structure where the protein must fold back on itself (so-called “turns”).
Amino Acids with Hydroxyl- or Sulfur-Containing Side Chains
In this category we can place serine, cysteine, threonine, methionine, and tyro-sine. Although methionine and tyrosine are fairly hydrophobic, these amino acids, because of their more polar side chains, display more hydrophilic charac-ter than their aliphatic analogs. As we will see in Chapcharac-ter 11, the group of serine and the group of cysteine are good nucleophiles and often play key roles in enzyme activity. Cysteine is noteworthy in two additional respects. First, the side chain can ionize at moderately high pH:
i SH i OH
a
a
a
142
CHAPTER 5 INTRODUCTION TO PROTEINS: THE PRIMARY LEVEL OF PROTEIN STRUCTURECOO− H3N C
H CH2 SH
+ H3N C COO− H+
H CH2 S−
+ +
pKa= 8.3
Second, oxidation of two cysteine side chains yields a disulfide bond:
oxidation reduction
COO− H3N C
H CH2 CH2
S S
+
C H
Cysteine Cystine
COO− H3N C
H CH2 SH
+
CH2 SH
−OOC C
H NH3+
2H+ + +2e−
−OOC NH3+
The variety of side chains on amino acids allows proteins enormous versatility in struc-ture and function.
AMINO ACIDS
143
The product of this oxidation is given the name cystine. We do not list it among the 20 amino acids because cystine is always formed by oxidation of two cysteine side chains and is not coded for by DNA. Such disulfide bonds often play an important role in stabilizing the structure of a protein.
Aromatic Amino Acids
Three amino acids, phenylalanine, tyrosine, and tryptophan, carry aromatic side chains. Phenylalanine, together with the aliphatic amino acids valine, leucine, and isoleucine, is one of the most hydrophobic amino acids. Tyrosine and tryptophan have some hydrophobic character as well, but it is tempered by the polar groups in their side chains. In addition, tyrosine can ionize at high pH:
O H
CH2 COO− H2N C
H
O−
CH2 COO− H2N C
H
pKa= 10.1
H+ +
10,000
1000
100
10 200 250 300
Wavelength, nm
Molar absorptivity, M–1cm–1 Tryptophan
Tyrosine
Phenylalanine
FIGURE 5.6
Absorption spectra of the aromatic amino acids in the near-ultraviolet region. Tryptophan (red; max
278 nm) and tyrosine (blue; max 274 nm) account for most of the UV absorbance by proteins in the region around 280 nm. Phenylalanine (black; max 258 nm) does not absorb at 280 nm. Note that the absorptivity scale is logarithmic. Compared with nucleic acids, amino acids absorb only weakly in the UV; see Figure 4.5 for comparison.
Reprinted from Advances in Protein Chemistry 17:303–390, D. B. Wetlaufer, Ultraviolet spectra of proteins and amino acids. © 1962, with permission from Elsevier.
l
l l
The aromatic amino acids, like most highly conjugated compounds, absorb light in the near-ultraviolet region of the spectrum (Figure 5.6). This characteristic is frequently used for the detection and/or quantitation of proteins, by measuring absorption at 280 nm.
Basic Amino Acids
Histidine, lysine, and arginine carry basic groups in their side chains. They are represented in Figure 5.3 in the form that predominates at pH 7. Histidine is the least basic of the three, and as its titration curve (Figure 5.7) shows, the imidazole ring in the side chain of the free amino acid loses its proton at about pH 6 ( values for the side chains of free amino acids are given in Table 5.1.). When histi-dine is incorporated into proteins, the typically ranges from 6.5–7.4 (Table 5.3). The value of for an ionizable side chain is sensitive to the proximity of other charged groups. In the folded structures of proteins the local electrostatic environment can perturb the of an ionizable side chain by three pH units.
Because the histidine side chain has a near physiological pH, it often plays a role in enzymatic catalysis involving proton transfer. Lysine and arginine are more basic amino acids, and as their values indicate (Tables 5.1 and 5.3), their side chains are almost always positively charged under physiological conditions. The guanidino group of arginine is a particularly strong base due to the resonance sta-bilization of the protonated side chain.
The basic amino acids are strongly polar, and as a consequence they are usu-ally found on the exterior surfaces of proteins, where they can be hydrated by the surrounding aqueous environment.
Acidic Amino Acids and Their Amides
Aspartic acid and glutamic acid typically carry negative charges at pH 7; they are depicted in the anionic forms in Figure 5.3. The titration curve of aspartic acid is shown in Figure 2.20 (page 46). The values of the acidic amino acids are so low (see Table 5.3) that even when the amino acids are incorporated into proteins, the negative charge on the side chain is typically retained under physiological conditions.
Hence, these amino acid residues are often referred to as aspartate and glutamate (i.e., the conjugate bases rather than the acids).
pKa pKa
pKa
; pKa
pKa
pKa
pKa
14 12 pH 10
8 6 4 2
00 1 2 3
Moles OH− added per mole histidine H2C H2N CH COO−
N HN
H2C H3N CH COO−
N HN
CH2 H3N CH COO−
HN H2C
CH COOH
NH HN
Histidine +
+
NH+ +
H3N+
FIGURE 5.7
Titration curve of histidine. The dots correspond to pKavalues, and the forms predominating at different pH values are shown. Ionizable hydrogens are shown in red.
It is presumed that the starting solution was adjusted to pH 2 by addition of Hto the dissolved amino acid.
See also Figure 2.20 (page 46) for the titration curve of aspartic acid.