Principles of Biochemistry Volume 1 - A. Lehninger 1985

Biomolecules
Proteins: Covalent Structure and Biological Functions
Chapter Summary

Proteins are the most abundant Class of macromolecules found within Cells, accounting for over half of their dry weight. They consist of very long polypeptide chains containing anywhere from 100 to 1,000 or more amino acid residues linked together by peptide bonds. Simple proteins yield only Amino Acids upon Hydrolysis, whereas Conjugated Proteins contain additional non-protein components, such as Metal Ions or organic prosthetic groups. Some proteins possess a fibrous (fibrillar) Structure and are insoluble, while others consist of tightly folded polypeptide chains and have a globular shape.

Cells contain hundreds and thousands of different proteins designed to perform A wide variety of biological Functions. Nevertheless, all of them are built from a set of the same 20 amino acids arranged in different sequences that are strictly defined for each specific protein. The Amino Acid Sequence of a polypeptide chain can be determined by cleaving it into smaller fragments and subsequently identifying The sequence of each fragment using Edman Degradation to remove N-terminal amino acid residues. The peptide fragments are then ordered correctly by identifying overlapping sequences among them. For this purpose, the original polypeptide is cleaved using a different method so that the new Cleavage sites do not coincide with those produced by the initial fragmentation. The Amino acid sequences of the second set of fragments must overlap the cleavage points obtained by the first method.

Homologous proteins isolated from different species exhibit Sequence Homology, meaning that the most critical positions in The polypeptide chains of homologous proteins are occupied by the exact same amino acids regardless of the Organism. At other positions, homologous proteins may contain different amino acids. The closer species are evolutionarily, the more similar The amino acid sequences of their homologous proteins. Thus, the sequences of homologous proteins indicate that the organisms containing them descended from a common ancestor, but underwent evolutionary divergence into distinct species. Similar Conclusions have been drawn from studies on antibody Specificity toward Antigens of homologous species.

When subjected to heating, extreme pH values, or certain chemical Reagents, Globular proteins typically become insoluble in Water and lose their biological activity while preserving the intact Covalent Structure of their polypeptide chain. This process, known as Denaturation, is caused by the unfolding of the polypeptide chains.

Introduction/47.html">Further Reading

Many useful insights can be found in the References for Chapter 5.

Books

Cooper T.G. The Tools of Biochemistry, Wiley, New York, 1977. A guide to experimental Methods in Protein Biochemistry. Dayhoff M.O. Atlas of Protein Sequence and Structure, vol. 5, suppl. 1-3, National Biomedical Research Foundation, Washington, D. C., 1972-1979. An encyclopedia of protein amino acid sequences. Dickerson R.E., Geis I. Proteins: Structure, Function, and Evolution, 2d ed., Benjamin/Cummings, Menlo Park, Calif., 1983. A beautifully illustrated book on proteins that serves as an excellent Introduction to the field.

Haschemeyer R., Haschemeyer A.N. Proteins: A Guide to Study by Physical and Chemical Methods. Wiley, New York, 1973.

Neurath H., Hill R.L. The Proteins, 3d ed., Academic, New York, 1977. A comprehensive and detailed multi-volume treatise on proteins.

Schultz G.E., Schirmer R.H. Principles of Cell/13.html">Protein Structure, Springer, New York, 1979.

Articles

Dickerson R.E. Structure and History of an Ancient Protein, Sci. Am., 226, 58-72, April (1972).

Edelman G.M. The Structure and function of Antibodies, Sci. Am., 223, 34-42, August (1970). Moore S., Stein W.H. Chemical Structures of Pancreatic Ribonuclease and Deoxyribonuclease, Science, 180, 458-464 (1973).

O'Farrell P.H. High Resolution Two-Dimensional Analysis of proteins, J. Biol. Chem., 250, 4007-4021 (1975). An interesting attempt to enumerate all proteins present in an E. coli cell.

Srinivasan P.R., Fruton J.S., Edsall J.T. (eds.) The Origins of Modern Biochemistry. A Retrospective on Proteins, Ann. N.Y. Acad. Sci., 325 (1979). A collection of fascinating articles on The history of protein research.

Questions and Problems

1. How many molecules of β-galactosidase are present in an E. coli cell? E. coli is a rod-shaped bacterium 2 µm long and 1 µm in diameter. When E. coli cells are grown on lactose (milk sugar) as a nutrient medium, the Bacteria synthesize the enzyme β-galactosidase (mol. wt. 450,000), which catalyzes the hydrolysis of lactose. The average density of a bacterial cell is 1.2 g/mL; soluble proteins account for 14% of the total cell mass, and β-galactosidase makes up 1.0% of these soluble proteins. Calculate the number of β-galactosidase molecules in an E. coli cell grown on lactose.

2. Number of Tryptophan residues in bovine serum albumin. According to quantitative Amino acid analysis, bovine serum albumin contains 0.58% (by weight) tryptophan, whose molecular weight is 204.

a) Calculate the minimum Molecular Weight of bovine serum albumin.

b) Gel filtration data indicate that the molecular weight of bovine serum albumin is approximately 70,000. How many tryptophan residues are present in a molecule of serum albumin?

3. Molecular weight of ribonuclease. The Lysine content of ribonuclease is 10.5% (by weight). Calculate the minimum molecular weight of ribonuclease. A ribonuclease molecule contains 10 lysine residues. Calculate the molecular weight of ribonuclease.

4. Net electrical charge of Polypeptides. A polypeptide isolated from the Brain has the sequence

Glu—His—Trp—Ser—Tyr—

Gly—Leu—Arg—Pro—Gly

Determine the net charge of the molecule at pH 3. What is its net charge at pH 5.5? At pH 8? At pH 11? The pK' values for the R-groups of Glu, His, Ser, Tyr, and Arg are 4.3, 6.0, 13.6, 10, and 12.48, respectively. Calculate the isoelectric point of this polypeptide.

5. Isoelectric point of Pepsin. Gastric juice pepsin (pH 1.5) has an isoelectric point of about 1, which is much lower than that of other proteins (see Table 6-5). Which functional groups must be present in pepsin in relatively large numbers for the enzyme to have such a low isoelectric point? Which amino acids contain these groups in their structure?

6. Isoelectric point of Histones. Histones are proteins found in the nuclei of Eukaryotic cells. They are tightly bound to deoxyribonucleic acid (DNA), which contains many phosphate groups. The isoelectric point of histones is very high (about 10.8). Which amino acid residues must be present in histones in relatively large amounts? How do these residues ensure the tight binding of histones to DNA?

7. Solubility of polypeptides. One method for separating polypeptides relies on differences in their solubility. As mentioned in the text, the solubility of large polypeptides in water depends on the polarity of their R-groups, particularly the number of ionizable groups: the greater the number of ionizable groups, the higher the solubility of the polypeptide. Which polypeptide in each of the following pairs is more soluble under the specified conditions?

a) (Gly)₂₀ or (Glu)₂₀ at pH 7.0;

б) (Lys—Ala)₃ or (Phe—Met)₃ at pH 7.0;

в) (Ala—Ser—Gly)₅ or (Asn—Ser—His)₅ at pH 9.0;

г) (Ala—Asp—Gly)₅ or (Asn—Ser—His)₅ at pH 3.0.

8. Cleavage of a polypeptide chain into fragments by Proteolytic Enzymes. Trypsin and Chymotrypsin are specific enzymes that catalyze the hydrolytic cleavage of polypeptides at specific sites in their chain (Table 6-6). The sequence of the B-chain of the polypeptide hormone Insulin is given below. Note that the cystine disulfide bridge between the A- and B-chains has already been cleaved by performic acid Treatment (see Fig. 6-12).

Phe — Val — Asn — Gln — His — Leu — CySO₃H — Gly — Ser — His — Leu — Val — Glu — Ala — Leu — Tyr — Leu — Val — CySO₃H — Gly — Glu — Arg — Gly — Phe — Phe — Tyr — Thr — Pro — Lys — Ala

Indicate the sites in the B-chain where cleavage occurs upon treatment with

a) trypsin, and b) chymotrypsin.

9. Determination of the amino acid sequence of leu-enkephalin, a brain-derived peptide. A group of polypeptides influencing Nerve Impulse transmission in specific brain regions was isolated from normal brain tissue. These polypeptides are known as opioids because they bind to specific receptors that also bind opiates (opium Alkaloids) such as morphine and naloxone. Thus, opioids mimic certain properties of opiates. Some researchers view these polypeptides as the brain's own pain-relief agents. Using the information provided below, determine the amino acid sequence of the opioid leu-enkephalin. Explain how the structure you derived is consistent with the following data:

a) Complete hydrolysis with 1 M HCl at 110°C followed by amino acid analysis revealed the presence of Gly, Leu, Phe, and Tyr in a molar ratio of 2:1:1:1.

б) Treatment of the polypeptide with 2,4-dinitrofluorobenzene followed by complete hydrolysis and chromatographic Separation of the products revealed the presence of a 2,4-dinitrophenyl derivative of Tyrosine. No free tyrosine was detected.

в) Partial hydrolysis of the polypeptide with chymotrypsin followed by chromatographic separation of the resulting products yielded Leu, Tyr, and a shorter peptide. Complete hydrolysis of the latter followed by amino acid analysis revealed the presence of Gly and Phe in a 2:1 ratio.

10. Electrophoresis of Peptides. When ionized Amino Acids and peptides are placed in an electric field, they migrate toward the cathode or anode depending on the pH (see Fig. 6-5). This method is widely used to separate peptides that differ in net charge. The method is particularly powerful because the net charge of a peptide can be altered by changing the pH of the medium.

a) Determine the direction of migration (toward the anode or cathode) for each of the amino acids and peptides listed below at the specified pH value:

1) Glu (pH 7)

2) Glu (pH 1)

3) Asp—His (pH 1)

4) Asp—His (pH 10)

б) At what pH value can the following three dipeptides—Gly—Lys, Asp—Val, and Ala—His—be easily separated using electrophoresis?

11. Solubility: salting-out

a) Pure proteins are largely insoluble in distilled water but dissolve in dilute salt solutions. However, when neutral salts are added in high concentrations to an aqueous protein solution, the protein precipitates out. This phenomenon is called salting-out. For example, most proteins dissolve in 0.1 M (NH₄)₂SO₄, but precipitate if the (NH₄)₂SO₄ concentration is increased to 3 M. After removing the excess (NH₄)₂SO₄ by dialysis, the proteins redissolve. Try to explain at THE MOLECULAR LEVEL why The addition of high salt concentrations leads to a decrease in Protein solubility.

b) The graph shows the dependence of the solubility of two proteins on the (NH₄)₂SO₄ concentration. How can these data be used to separate proteins A and B?

Problem 11

12. Affinity Chromatography: A highly specific and efficient method for isolating specific proteins. Because the molecules of most proteins are easily degraded when subjected to many of the traditional Isolation and Purification techniques used in organic chemistry—such as sublimation and extraction with various Solvents—biochemists have been forced to develop specialized methods for this purpose. It is frequently possible to isolate a single protein present in a concentration of 10^-3–10^-6 M from a mixture containing several thousand other Biomolecules. One such method, known as affinity chromatography, has played a decisive role in the isolation and purification of A number of enzymes, IMMUNOGLOBULINS, and receptor proteins. The method is based on the well-established fact that proteins, in carrying out their biological functions, reversibly bind to other specific types of molecules called ligands. This results in The formation of stable, non-covalent protein-Ligand complexes.

In this method, the ligand that specifically binds the protein of interest is covalently attached to insoluble polymer beads 10–50 µm in diameter.

To isolate the protein from a cell extract, the extract is applied to a Column packed with the polymer beads carrying the attached ligand, and the column is then washed repeatedly with a buffer solution. Only those proteins with a high affinity for the immobilized ligand are retained on the column, whereas the other proteins are simply washed through by the buffer. Because the affinity and Specificity of the protein for the ligand are very high, this approach can often isolate and purify extremely small amounts of a protein in a single step from a cell extract containing hundreds of other proteins.

How can the protein remaining on the affinity column be recovered in pure form? Explain the principles underlying this Procedure.

13. Structure of a polypeptide antibiotic isolated from Bacillus brevis. Extracts obtained from a bacterial culture of Bacillus brevis contain a peptide with antibiotic properties. This peptide forms complexes with metal ions and apparently disrupts the ion transport system across The Cell membrane, thereby killing certain species of bacteria. The structure of the polypeptide was determined based on the following observations:

a) Complete acid hydrolysis of the peptide, as shown by subsequent amino acid analysis, yielded equimolar amounts of Leu, Orn, Phe, Pro, and Val. Orn is the abbreviation for Ornithine, an amino acid not found in proteins but present in certain peptides. It has the following structure:

b) Molecular weight measurements gave an approximate value of 1200.

c) The peptide is not susceptible to hydrolysis by the enzyme carboxypeptidase.

d) Treatment of the original polypeptide with fluorodinitrobenzene, followed by complete hydrolysis of the resulting derivative and chromatography of the products, yields only free amino acids and a derivative of the following structure:

(Hint: Note that the 2,4-dinitrophenyl group has attached not to the α-nitrogen, as is usually the case, but to the side-chain amino group.)

e) Partial hydrolysis of the polypeptide, followed by chromatographic separation of the products and determination of their amino acid sequences, yielded the following di- and tripeptides (the N-terminal amino acid is always on the left):

Based on the information above, try to deduce the amino acid sequence of the polypeptide antibiotic. Explain your reasoning. Once you have your answer, go back and check how well the structure you determined is consistent with each of the observations described above.