Amino Acids, Peptides And Proteins

Non-polar R Groups

Eight amino acids are classified as having non-polar side chains (R group). In proteins these amino acid residues are usually buried in the hydrophobic interior of the biomolecule and are out of contact with water. Alanine, valine, leucine and isoleucine have aliphatic hydrocarbon side chains ranging in size from methyl group for alanine to isomeric butyl groups for leucine and isoleucine (Fig. 4.4 ). Alanine is the most abundant amino acid in most proteins. Phenylalanine with its phenyl moiety and tryptophan with its indole group contain aromatic side chains, and (together with the aliphatic amino acids) contribute to the internal hydrophobic interactions of the proteins. They are also responsible for the ultraviolet absorption of most proteins, discussed later. Methionine has a thiol ether side chain. Proline differs from other amino acids in that its side chain pyrrolidine ring includes both the α-carbon and the α-amino group. Chemically speaking proline is not an α-amino (–NH₂) acid but rather an α-imino (–NH) acid.

Uncharged Polar R Groups

Side chains of these amino acids are uncharged, and they have polar groups (–OH, –SH, –NH, C = O) that can hydrogen bond to water. Serine and threonine, for example, bear hydroxylic groups of different sizes and glycine has hydrogen for its R group. Asparagine and glutamine have amide-bearing side chains of different sizes. Tyrosine has a phenolic group (and like phenylalanine and tryptophan is aromatic). Cysteine has a thiol group that can form disulphide bond with another cysteine through oxidation of the two thiol groups to form cystine (Fig. 4.5 ).

Charged Polar R Groups

Five amino acids have charged side chains (Fig. 4.4), having acidic or basic groups. These groups can assume respectively, negative or positive charge at physiologic pH values.

The allocation of the 20 amino acids among the three different subgroups is somewhat arbitrary. The following examples illustrate this point:

• Aliphatic amino acids:

    Branched chain amino acids: Valine, leucine, isoleucine Sulphur containing amino acids: Methionine, cysteine Amide group containing amino acids: Asparagine, glutamine Hydroxy amino acids: Serine, threonine, tyrosine Simple amino acids: Glycine and alanine

• Aromatic amino acids: Phenylalanine, tyrosine, tryptophan

• Imino acid: Proline

• Heterocyclic amino acids: Histidine, tryptophan

• Dicarboxylic amino acids: Aspartic acid and glutamic acid

• Dibasic amino acids: Arginine, lysine

1. The hydroxyl groups of serine and threonine exist at the catalytic sites of certain kinds of enzyme, e.g. the enzymes that regulate energy metabolism and fuel storage in the body. They can be phosphorylated, hence their charge changes from neutral to negative, which changes the shape and function of the enzyme proteins. Thus, these (hydroxyl) groups are important in the regulation of activities of these enzymes (Chapter 5).

2. Thiol group of a cysteine can form a disulphide bond with another cysteine through the oxidation of the two thiol groups (Fig. 4.5). The dimeric compound so formed, called cystine, is important in cross-linking the adjacent polypeptide chains.

3. Amide bearing side chain of asparagine forms covalent linkage with oligosaccharide unit (called N-glycosidic linkage) in glycoproteins. The –OH groups of serine and threonine also similarly form linkage with reducing end of oligosaccharide, called O-glycosidic linkage (Chapter 5).

4. Aromatic side chains are responsible for the ultraviolet absorption of most proteins, which have absorption maxima between 275 nm and 285 nm. Tryptophan has greater absorption in this region than the other two aromatic amino acids. Since nearly all proteins contain aromatic amino acids, the amount of light absorbed at 280 nm by a protein is used as an indirect measure of protein concentration.

5. Hydroxyl group of tyrosine is subject to phosphorylation in some enzymes, which is important in the regulation of metabolic pathways.

6. Imidazole group of histidine is important in the buffering activities of proteins.

7. Cyclic pyrrolidine group of proline introduces bend in the peptide chain.

8. Butylammonium side chain of lysine binds with the co-enzymes pyridoxal phosphate and biotin.

9. R group of glycine provides little stearic hindrance because of its small size, so that proteins can bend or rotate easily wherever glycine forms part of their structure.

1. Carboxyl group, which can donate a proton, therefore acting as an acidic group.

2. Amino group, which is capable of accepting a proton, thereby acting as a basic group.

1. Isoelectric pH of the amino acid.

2. pH of the surrounding medium.

Titration Curve of a Monoamino-monocarboxylic Acid (Alanine)

The titration curve of alanine proceeds in the same way as that of a weak acid. There is, however, an important difference: the titration curve of this amino acid proceeds in two distinct stages. This is because an amino acid molecule, when fully protonated, is capable of donating two protons. Each of the two stages of the titration corresponds to loss of a proton (Fig. 4.7 ). In the first stage of titration, dissociation of the carboxyl group occurs while the second stage involves dissociation of the amino group.

Stage I: Dissociation of the carboxyl group: At low pH both the carboxyl group as well as the amino group of the amino acid are protonated, and hence, the predominant ionic species is the diprotonated (positive) form. As the pH is raised by addition of NaOH, the COOH group donates its proton to neutralize the hydroxyl group of the alkali (i.e. sodium hydroxide). Thus, formation of the carboxyl (COO^-) ion occurs at the expense of COOH group, so that some diprotonated form is converted to the dipolar (zwitterion) form. At the end of this stage of titration, dissociation of carboxylate group is complete and the diprotonated form is almost completely converted to the dipolar (Z) form.

Midway through this stage, the following relations exist:

The solution has buffering power in this zone, as shown by flat shape of the titration curve. Normally, a system acts as buffer in the pH zone of pK' ± 1. Since pK' of the carboxyl group (pK’₁ of alanine is 2.34, it acts as a buffer in the pH zone of2.34 ± 1 (i.e. from pH range of1.34 to 3.34).

Stage II: Dissociation of the amino group: Dissociation of proton from the amino group of alanine occurs in this stage, resulting in the formation of the deprotonated (negative) form from the Z form (Fig. 4.11 ).

Midway through this stage, dissociation of amino group is half complete, and the following relations exist:

The solution has buffering power at this stage as evidenced by a flat zone in the titration curve. Since pK' of the amino group is 9.69, the second buffering zone of alanine lies in the pH zone of 9.69 ± 1 (i.e. from 8.69 to 10.69).

The following observations about the titration curve are noteworthy.

where, pK’₁ and pK’₂ = pK' values of α-carboxyl and the α-amino groups respectively, pI = isoelectric pH.

At this pH, both the amino group and the carboxyl groups of the amino acid are ionized, but the overall charge on the molecule in nil.

Titration Curve of Basic Amino Acids

These amino acids have a third ionizable amino, group in side chain. Since this group can reversibly gain or lose a proton, the basic amino acids have an additional third phase of titration. Accordingly, there are three regions of buffering; one region each corresponding to loss of a proton from the carboxyl group, the amino group, and the side–CHain. The pK' value of the imidazole group of histidine is 6.0, and therefore, it can serve as a buffer in pH range of 6.0 ± 1, which is very close to the physiological pH. Hence, histidine has the capacity to provide buffering action at the body pH.

Titration Curve of Acidic Amino Acids

These amino acids have a third dissociable group, i.e. the carboxyl group in side chain. Their titration curves, therefore, have an additional third stage corresponding to this carboxyl group.

• Not only the bond length, but strength of peptide bonds also lies half-way: stronger than single bond and weaker than double bond. a consequence of the partial double bond character is that there is lack of rotational freedom, discussed above.

φ and Angles

In addition to the peptide bond, the polypeptide backbone contains bonds between the a₁-carbon and the peptide bond carbon, and between the α-carbon and the peptide bond nitrogen. These two being single bonds show free rotation. Rotation around the nitrogen-and-α- carbon bond is measured as the φ (phi) angle and rotation around the peptide carbon and α-carbon bond as the ψ (psi) angle.

Fibrous Proteins

The polypeptide chains of these proteins extend along a longitudinal axis without showing any sharp bends of folding (Fig. 4.11). Simplicity of structure of these proteins is responsible for their mechanical properties. They are essential components of several proteins, including collagen and elastin (for details see Chapter 5).

Globular Proteins

Polypeptide chain of a globular protein is tightly folded and packed into a compact structure (Fig. 4.11). The hydrophilic groups of the constituent amino acids are exposed to the exterior, so that these proteins can diffuse through aqueous systems. Most enzymes, transport proteins, nutrient proteins, antibodies and hormones are globular.

Simple Proteins

Simple proteins comprise solely of amino acids; no other chemical group is present in them. Pancreatic ribonuclease is an example.

Conjugated Proteins

Conjugated proteins contain a polypeptide group as well as a non-polypeptide group. In these proteins, the non-polypeptide group is called the prosthetic group, and the polypeptide is called the apoprotein. a prosthetic group is often employed when the biological function of the protein requires a functional group that is not available in any of the 20 amino acids. For example, in oxygen transport proteins, haemoglobin and myoglobin, binding of oxygen occurs with the iron-porphyrin group. The prosthetic group is sugar in glycoproteins (Greek: glycos means sweet), lipid in lipoproteins, and a metal in metalloproteins (Table 4.7 ).

α-Helix

It is a spiral structure consisting of a polypeptide chain that is coiled around a longitudinal axis in a helical fashion (Fig. 4.14a ). The α-helical structure was first defined by Linus Pauling from his studies on fibrous proteins. However, short stretches of α-helix are found in the globular proteins as well. a vast variety of proteins contain the α-helical structure. They constitute almost the entire dry weight of hair, wool, feathers, and scales and form much of the outer layer of skin.

β-Pleated Sheet

In 1951, the same year Pauling proposed α-helix, Pauling and Corney reported existence of a different type of secondary structure, the β sheet.

Folding of Proteins and Chaperones

Polypeptide chain of a given amino acid sequence (i.e. primary structure) always folds in the same characteristic fashion, suggesting that the information for protein folding is inherent in the primary structure itself. This is in line with the Anfinsens law stating that the secondary, tertiary and quaternary structures are determined by the primary structure. indeed, protein folding starts, and often makes considerable headway, while translation is still in progress (Chapter 22).

In addition to the primary structure, certain other factors also determine the overall folding pattern. This is evident from the fact that in some cases, following denαturation of protein (i.e. unfolding) by temperature or pH alterations, the native conformation is not regained (by the unfolded protein) even when the original temperature or pH is restored.

It is now known that a group of specialized proteins called “chaperones” are required for the native folding of many proteins. The chaperones are also called polypeptide chain binding (PCB) proteins. Though they aid in folding during synthesis, they are not responsible for the stability of the final product. Chaperones bind to hydrophobic patches and stabilize intermediates during folding. They prevent aggregation of incompletely folded proteins by precluding adventitious contacts between exposed hydrophobic regions. Defective interaction of a polypeptide with the corresponding chaperone may cause disease (Case 7.2).

The energy requirements of chaperones can be substantial, and more than 120 ATP molecules can be expended during the folding of a single polypeptide chain. Many chaperones are heat shock or stress proteins, but they primarily serve the aforementioned function under physiological conditions.

Domains

Long polypeptides that contain more than 200 residues fold into two or more globular clusters known as domains. The domains are connected by more extended part of the polypeptide chain, which gives these polypeptides a bior multi-lobal appearance. Each subunit of the enzyme glyceraldehyde 3-phosphate dehydrogenase, for example, has two domains connected by more extended part of the polypeptide chain.

Many domains are structurally independent units, each having characteristics of a small globular protein. Generally, domains are formed from between 40 to 400 amino acid residues (mean size = 200 amino acid residues). Different domains may have different biological functions.

Forces Stabilizing the Tertiary Structure

Tertiary structure mostly represents a state of lowest energy and imparts greatest stability for the polypeptide in question. Five kinds of interactions, cooperate to stabilize it (Fig. 4.17 ). These are:

1. Solubility: Water-soluble proteins become insoluble, the boiling of an egg is a familiar example of this phenomenon.

2. Precipitation: As the protein becomes less symmetric, it exerts resistance to various types of movements, and in many cases it precipitates.

3. Optical rotation: a more negative optical rotation results specific rotation of native proteins is between —20° and —40°, whereas that of denatured proteins is near –100°.

4. The sedimentation rate and diffusion rate fall and the intrinsic viscosity increases.

1. Heat: Most proteins are denatured at temperatures between 50°C and 80°C; at these temperatures the non-covalent interactions, which are individually very weak, are readily broken.

2. Detergents and organic solvents: They serve to disrupt hydrophobic interactions. Being non-polar, they insert themselves between the side chains of hydrophobic amino acids.

3. Strong acids and bases: They disrupt the intramolecular salt bonds by changing the charge pattern of the protein. in a strong acid, the protein loses its negative charges; and in a strong base, it loses its positive charges; both deprive the protein of the salt bonds.

4. 8M Urea or 6M guanidine hydrochloride: Both are hydrophilic agents with high hydrogen binding potentials. They can denature proteins by disrupting the hydrogen bonds between the protein and the surrounding water molecules. Normally the water molecules assume an “ordered” position at an aqueous/ non-polar interface, which is reduced by these agents, and consequently the hydrophobic interactions within the protein are weakened.

5. Heavy metal ions: They have high affinity for carboxylate groups, and for the sulphydryl groups in many proteins.

6. Trichloroacetic acid (TCA): It is of special importance in laboratories because it can rapidly denature and precipitate proteins, which can then be removed by centrifugation.

7. Physical agents: X-Rays, ultraviolet rays, high pressure, vigorous shaking and surface tension also cause denaturation.

(i) Salting-in: It is shown in Figure 4.18b . The protein molecules have multiple salt bonds among them, which cause them to aggregate. As a result they are insoluble (Fig. 4.18a). In 5% NaCl, salt ions bind to surface charges of protein molecules, thereby preventing intermolecular salt bonds. This causes increased solubility.

    Thus higher solubility at a moderate concentration (1–10% salt) is because the salt ions shield the protein’s multiple ionic charges, thereby weakening the attractive forces between different protein molecules.

(ii) Salting-out: Excess salt, however, precipitates the proteins because most of the water molecules become tied up in forming the hydration shells around the salt ions. Effectively, so many salt ions are hydrated that there is significantly less bulk water available to dissolve other substances, including proteins. Ammonium chloride, one of the most soluble salts, is the most commonly used reagent for salting out the plasma proteins.

Gel Filtration (Column Chromatography)

Particles of different sizes can be separated from one another by gel filtration, also called, size-exclusion or molecular-sieve chromatography. The equipment consists of a column, which is packed with a beaded hydrophilic material such as Sephadex gel. Sephadex is a dextran derivative, obtained from bacterial cell walls. a gel bead contains numerous pores. The pore size is such that the smaller proteins can penetrate the porous gel beads, while the larger proteins bypass the beads altogether (Fig. 4.21 ). Having access to gels internal volume, the small proteins take a longer and tortuous route through the column. Therefore, they are retarded on the column with respect to the larger protein that cannot enter the porous gel beads. Thus, the latter traverse the column more rapidly and emerge earlier in the column effluent than the smaller ones. The proteins of intermediate size have partial access to gel’s internal volume, and therefore, emerge in between, before the larger proteins but after the smaller proteins.

Fractions of a few milliliters (each fraction called an elute) are collected from the bottom of the column; the process is called elution. Each of these elutes would contain different size particle.

Ultracentrifuge

Proteins of different molecular sizes can be separated by high-speed centrifugation into different components. Rate at which a protein moves in a centrifugal field (i.e. sedimentation) depends on its size; larger proteins sediment faster than the smaller ones. This method can separate those proteins which have large differences in their molecular size.

Ion-exchange Chromatography

Preparative separation of proteins can be achieved on the basis of charge-differences by this technique. The method takes advantage of differential affinity between the following:

The resins are packed in chromatography column (Fig. 4.22 ). They are cross-linked polymers (e.g. cellulose or agarose) containing charged groups as part of their structure. The charged groups most commonly attached are diethylaminoethyl (DEAE) group and carboxymethyl (CM) group:

CM: Resins–CH₂COO^- (Negative)

DEAE groups are protonated and have a positive charge associated with a negatively charged counterion, such as chloride; this system is called DEAE-cellulose chromatography (Fig. 4.23 ). If a protein solution at a specific pH is applied to this column:

CM groups with negative charge are associated with counterion Na⁺. (Fig. 4.24 ). In this case, positively charged proteins exchange with Na⁺ and get bound to the resin (i.e. cation exchange), whereas the negative ones are eluted faster.

High Performance Liquid Chromatography (HPLC)

HPLC resembles the conventional chromatographic techniques where the solution of proteins is passed through special resins that have attached side groups. These side groups can react with the protein sample ionically or hydrophobically. However, there is one important differences HPLC is carried out under high pressure (5000–10,000 pounds per square inch) in the chromatographic matrix. The matrix consists of specially fabricated 3–300 μm-diameter glass or plastic beads, which are coated with a uniform layer of chromatographic material. This greatly improves the speed, resolution, and reproducibility of the separation.

Electrophoresis

This process uses an electric field to drive the movement of charged particles. It is used for the separation of a wide variety of polyelectrolytes (carry positive and negative charges), including amino acids, polypeptides, proteins (and DNA).

At pH values greater than the isoelectric point (pI), the protein carries an excess of negative charge and moves to the anode; at pH values less than pi, it carries positive charge and moves towards the cathode; and at isoelectric point the net charge is zero and it does not move. if for example, an alkaline pH is used (greater than pI of most proteins) the serum proteins are negatively charged and move towards the anode, but are separated according to their net charge (Fig. 4.25 ). a porous support such as paper, cellulose acetate foil, starch gel, or other carrier material is commonly used to minimize diffusion and convection. Electrophoresis is the most common method of protein separation in the clinical laboratory, used for separation of plasma proteins and isoenzymes and for the detection of structurally abnormal proteins. Case 5.1 illustrates diagnostic utility of electrophoresis in a bone disorder.

Affinity Chromatography

This is the only method of protein purification that exploits a property related to the function of the protein, namely the ligand-binding ability. This is a definite advantage over exploiting small differences in the physicochemical properties between proteins in the other chromatographic methods.

The column is packed with a chromatographic material that carries an immobilized ligand (Fig. 4.27 ).

The mixture of proteins is poured over the column. As it percolates down, the protein molecules with an appropriate ligand-binding site gets bound to the immobilized ligand, whereas the other proteins are washed through the column with the buffer. The bound protein can then be recovered in highly purified form by changing the elution conditions. (Generally, it is washed with solution having a high concentration of soluble-ligands.)

Precipitation by Antibodies

For separating a given protein from a protein mixture, antibodies are prepared against it. When these antibodies are added to the protein mixture, they form antigenantibodies complexes with protein of interest. These complexes are large in size, and so can be separated from the rest of mixture by centrifugation. Since it is necessary to create complexes of sufficiently large size, the antiglobulin (anti IgG) is further added, so that the large triple complex (of antigen-antibody-anti IgG) is formed.

Step 1: The size of the protein

The molecular size of the protein is estimated usually by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), as discussed earlier. Since all proteins are covered with negative charges of SDS, they tend to be linear in shape and their movement in electric field solely depends on their size.

Step 2: Amino acid composition of the protein

It is determined by complete hydrolysis of the polypeptide, followed by the analysis of the liberated amino acids. The hydrolysis is accomplished by acid hydrolysis of the peptide linkages in 6N–HCl for 24–72 hours at 100°–110°C. This treatment, however, destroys tryptophan. Alkaline hydrolysis is, therefore, used to liberate this amino acid. The amino acids so liberated are then separated by chromatographic methods and determined quantitatively with suitable reagents. Modern amino acid analyzers can completely analyze a protein digest containing as little as 1 pmol of each amino acid in less than an hour.

Step 2 reveals the protein’s amino acid composition, but not the amino acid sequence. it is, however, useful because the ratio of the amino acids can be compared with that from known proteins and this may give a clue to its structure.

Step 3: Determination of N and C terminal amino acids

The N-terminal amino acid is covalently bound to dansyl chloride. The tagged protein is then hydrolyzed to its constituent amino acids by acid hydrolysis. Because only the N-terminal amino acid is dansylated, it is identified by separating it out by column chromatography and comparing against dansylated standards.

The C-terminal amino acid is determined by treating intact polypeptide with hydrazine, which reacts hydrolytically at the carbonyl grouping (C–O) at each peptide bond (resulting in acyl derivatives of each residue). However, it cannot react with the C-terminal, which has carboxyl group (rather than a peptide linkage). The amino acids are then analyzed by column chromatography and compared with standards. The amino acid without hydrazine is the C-terminal amino acid.

Step 4: Sequence analysis

It is performed by Edman degradation method on automatic machines called, cyclic sequenators. This method sequentially removes one residue at a time from the amino end of a peptide. Phenyliosthiocyanate reacts with the N-terminal amino group of the peptide to form a phenylthiocarbamyl derivative in alkaline solution. It is released by gentle acid hydrolysis and identified by HPLC. The second amino acid now becomes the N-terminal amino acid and the cycle can be repeated by returning to alkaline conditions.

Only about 20–25 amino acids can be sequenced in this way. The large polypeptides, however, must be fragmented first. a number of fragmenting agents break specific peptide linkages, among which are digestive endopeptidases, trypsin and chymotrypsin.

Step 5: Reconstructing the protein’s sequence

After individual peptide fragments have been sequenced, their order in the original polypeptide must be elucidated. For this a second round of protein cleavage with a different endopeptidase is carried out. This generates an overlapping set of peptide fragments. These fragments are then separated and sequenced. The sequences of the overlapping peptides are then used, much as one would fit parts of a jigsaw puzzle, to obtain the whole protein structure.

Step 1

The protein is partially purified and the first eight (or ten) amino acids at the N-terminal end are sequenced using an automatic sequenator.

Step 2

These eight amino acids are compared with an international protein/DNA sequence library available on the Internet, e.g. GenBank. If lucky, a match occurs in terms of molecular weight, which means that the protein (or a closely similar protein) has already been isolated and sequenced. Further sequencing may not be necessary in this case.

Step 3

If no match is found, then a cDNA molecule that codes for the N-terminal amino acid sequence is made. This is called cDNA primer.

Step 4

Using the cDNA as a probe, the RNA that codes for the protein is extracted from the cells. The two readily hybridize (attach together) because their sequences are complementary.

Step 5

The DNA complementary to the rest of the RNA sequence is then synthesized by polymerase chain reaction (Chapter 25). This results in the synthesis of large amounts of DNA that codes for the protein.

Step 6

The DNA is then sequenced using techniques described in Chapter 21 and, using the universal genetic code, and the protein sequence is derived from the DNA sequence.

This process sounds complicated but offers advantage that very little of the protein is needed and the protein does not have to be fully purified. Full purification is especially difficult in case of insoluble proteins (e.g. membrane proteins). Moreover, sequencing DNA is a relatively simple procedure.

Ultraviolet Light Spectroscopy

Proteins absorb ultraviolet radiation with two absorption maxima: one at 190 nm is caused by peptide bonds, and the other at 280 nm is caused by the aromatic side chains of tyrosine and tryptophan (aromatic character of purine and pyrimidine bases also causes absorbance peak at 260 nm for nucleic acids).

Nuclear Magnetic Resonance (NMR)

Since mid 1980s advances in NMR spectroscopy have permitted the determination of the three-dimensional structure of proteins (and nucleic acids) in aqueous solution. The sample of protein is placed in a magnetic field so that the spin of protons is aligned. Radiofrequency pulses are applied, which cause excitation of protons. The excited protons then emit signals, whose frequency depends on the molecular environment of these protons. interpretation of these signals helps to construct the three-dimensional protein structure. The technique is useful for the proteins that fail to crystallize. However, utility of this technique is limited to a size of less than 30 kD.

Computer Based Modelling

A number of proteins have been modelled till now, and based on them computer programs have been developed to predict the most likely tertiary structure from the primary amino acid sequence.

1. What do you mean by the term conformation as applied to a protein? Describe the various orders of structure of proteins.

2. Define secondary and tertiary structures. Mention the bonds that are responsible for maintaining these structures.

3. Give two examples each of fibrous and globular proteins. Describe the structural and functional relationship with respect to any one protein.

4. Describe the separation techniques that exploit the following molecular properties: charge, polarity, size and specificity.

5. Why are proteins called amphoteric molecules? Explain why histidine, but not alanine, can act as buffer at physiologic pH.

1. Quaternary structure

2. α-Helix

3. β-Pleated sheets

4. Essential amino acids

5. Chaperones

6. isoelectric pH

7. Gel chromatography

8. Edman degradation

9. Titration curve of histidine

10. Physiologically active peptides

11. Electrophoresis