Valuable reagents. The following reagents are often used in protein chemistry:

Which one is the best suited for accomplishing each of the following tasks?

The only constant is change. Explain how two different cell types from the same organism will have identical genomes but may have vastly divergent proteomes.

Crafting a new breakpoint. Ethyleneimine reacts with cysteine side chains in proteins to form S-aminoethyl derivatives. The peptide bonds on the carboxyl side of these modified cysteine residues are susceptible to hydrolysis by trypsin. Why?

Spectrometry. The absorbance A of a solution is defined as

A = log₁₀(I₀/I)

in which I₀ is the incident-light intensity and I is the transmitted-light intensity. The absorbance is related to the molar absorption coefficient (extinction coefficient) ε (in M⁻¹ cm⁻¹), concentration c (in M), and path length l (in cm) by

A = εlc

The absorption coefficient of myoglobin at 580 nm is 15,000 M ⁻¹ cm⁻¹. What is the absorbance of a 1 mg ml⁻¹ solution across a 1-cm path? What percentage of the incident light is transmitted by this solution?

It’s in the bag. Suppose that you precipitate a protein with 1 M(NH₄)₂SO₄ and that you wish to reduce the concentration of the (NH₄)₂SO₄. You take 1 ml of your sample and dialyze it in 1000 ml of buffer. At the end of dialysis, what is the concentration of (NH₄)₂SO₄ in your sample? How could you further lower the (NH₄)₂SO₄ concentration?

Too much or not enough. Why do proteins precipitate at high salt concentrations? Although many proteins precipitate at high salt concentrations, some proteins require salt to dissolve in water. Explain why some proteins require salt to dissolve.

A slow mover. Tropomyosin, a 70-kDa muscle protein, sediments more slowly than does hemoglobin (65 kDa). Their sedimentation coefficients are 2.6S and 4.31S, respectively. Which structural feature of tropomyosin accounts for its slow sedimentation?

Sedimenting spheres. What is the dependence of the sedimentation coefficient s of a spherical protein on its mass? How much more rapidly does an 80-kDa protein sediment than does a 40-kDa protein?

Frequently used in shampoos. The detergent sodium dodecyl sulfate (SDS) denatures proteins. Suggest how SDS destroys protein structure.

Size estimate. The relative electrophoretic mobilities of a 30-kDa protein and a 92-kDa protein used as standards on an SDS–polyacrylamide gel are 0.80 and 0.41, respectively. What is the apparent mass of a protein having a mobility of 0.62 on this gel?

Unexpected migration. Some proteins migrate anomalously in SDS-PAGE gels. For instance, the molecular weight determined from an SDS-PAGE gel is sometimes very different from the molecular weight determined from the amino acid sequence. Suggest an explanation for this discrepancy.

Sorting cells. Fluorescence-activated cell sorting (FACS) is a powerful technique for separating cells according to their content of particular molecules. For example, a fluorescence-labeled antibody specific for a cell-surface protein can be used to detect cells containing such a molecule. Suppose that you want to isolate cells that possess a receptor enabling them to detect bacterial degradation products. However, you do not yet have an antibody directed against this receptor. Which fluorescence-labeled molecule would you prepare to identify such cells?

Column choice. (a) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Which method would be most appropriate for separating the products: ion-exchange or gel-filtration chromatography? Explain. (b) Suppose that the peptide was digested with chymotrypsin. What would be the optimal separation technique? Explain.

Power(ful) tools. Monoclonal antibodies can be conjugated to an insoluble support by chemical methods. Explain how these antibody-bound beads can be exploited for protein purification.

Assay development. You wish to isolate an enzyme from its native source and need a method for measuring its activity throughout the purification. However, neither the substrate nor the product of the enzyme-catalyzed reaction can be detected by spectroscopy. You discover that the product of the reaction is highly antigenic when injected into mice. Propose a strategy to develop a suitable assay for this enzyme.

Making more enzyme? In the course of purifying an enzyme, a researcher performs a purification step that results in an increase in the total activity to a value greater than that present in the original crude extract. Explain how the amount of total activity might increase.

Divide and conquer. The determination of the mass of a protein by mass spectrometry often does not allow its unique identification among possible proteins within a complete proteome, but determination of the masses of all fragments produced by digestion with trypsin almost always allows unique identification. Explain.

Know your limits. Which two amino acids are indistinguishable in peptide sequencing by the tandem mass spectrometry method described in this chapter and why?

Protein purification problem. Complete the following table.

Part of the mix. Your frustrated colleague hands you a mixture of four proteins with the following properties:

(a) Propose a method for the isolation of Protein B from the other proteins. (b) If Protein B also carried a His tag at its N-terminus, how could you revise your method?

The challenge of flexibility. Structures of proteins comprising domains separated by flexible linker regions can be quite difficult to solve by x-ray crystallographic methods. Why might this be the case? What are possible experimental approaches to circumvent this barrier?

Quaternary structure. A protein was purified to homogeneity. Determination of the mass by gel-filtration chromatography yields 60 kDa. Chromatography in the presence of 6 M urea yields a 30-kDa species. When the chromatography is repeated in the presence of 6 M urea and 10 mM β-mercaptoethanol, a single molecular species of 15 kDa results. Describe the structure of the molecule.

Helix–coil transitions. (a) NMR measurements have shown that poly-l-lysine is a random coil at pH 7 but becomes a helix as the pH is raised above 10. Account for this pH-dependent conformational transition. (b) Predict the pH dependence of the helix–coil transition of poly-l-glutamate.

Peptide mass determination. You have isolated a protein from the bacterium E. coli and seek to confirm its identity by trypsin digestion and mass spectrometry. Determination of the masses of several peptide fragments has enabled you to deduce the identity of the protein. However, there is a discrepancy with one of the peptide fragments, which you believe should have the sequence MLNSFK and an (M+H)⁺ value of 739.38. In your experiments, you repeatedly obtain an (M+H)⁺ value of 767.38. What is the cause of this discrepancy and what does it tell you about the region of the protein from which this peptide is derived?

Peptides on a chip. Large numbers of different peptides can be synthesized in a small area on a solid support. This high-density array can then be probed with a fluorescence-labeled protein to find out which peptides are recognized. The binding of an antibody to an array of 1024 different peptides occupying a total area the size of a thumbnail is shown in the adjoining illustration. How would you synthesize such a peptide array? (Hint: Use light instead of acid to deprotect the terminal amino group in each round of synthesis.)

Exchange rate. The amide hydrogen atoms of peptide bonds within proteins can exchange with protons in the solvent. In general, amide hydrogen atoms in buried regions of proteins and protein complexes exchange more slowly than those on the solvent-accessible surface do. Determination of these rates can be used to explore the protein-folding reaction, probe the tertiary structure of proteins, and identify the regions of protein–protein interfaces. These exchange reactions can be followed by studying the behavior of the protein in solvent that has been labeled with deuterium (²H), a stable isotope of hydrogen. What two methods described in this chapter could be readily applied to the study of hydrogen–deuterium exchange rates in proteins?

Protein sequencing 1. Determine the sequence of hexapeptide on the basis of the following data. Note: When the sequence is not known, a comma separates the amino acids (Table 3.3).

Amino acid composition: (2R,A,S,V,Y)

N-terminal analysis of the hexapeptide: A

Trypsin digestion: (R,A,V) and (R,S,Y)

Carboxypeptidase digestion: No digestion.

Chymotrypsin digestion: (A,R,V,Y) and (R,S)

Protein sequencing 2. Determine the sequence of a peptide consisting of 14 amino acids on the basis of the following data.

Amino acid composition: (4S,2L,F,G,I,K,M,T,W,Y)

N-terminal analysis: S

Carboxypeptidase digestion: L

Trypsin digestion: (3S,2L,F,I,M,T,W) (G,K,S,Y)

Chymotrypsin digestion: (F,I,S) (G,K,L) (L,S) (M,T) (S,W) (S,Y)

N-terminal analysis of (F,I,S) peptide: S

Cyanogen bromide treatment: (2S,F,G,I,K,L,M*,T,Y) (2S,L,W)

M*, methionine detected as homoserine

Applications of two-dimensional electrophoresis. Performic acid cleaves the disulfide linkage of cystine and converts the sulfhydryl groups into cysteic acid residues, which are then no longer capable of disulfide-bond formation.

Consider the following experiment: You suspect that a protein containing three cysteine residues has a single disulfide bond. You digest the protein with trypsin and subject the mixture to electrophoresis along one end of a sheet of paper. After treating the paper with performic acid, you subject the sheet to electrophoresis in the perpendicular direction and stain it with a reagent that detects proteins. How would the paper appear if the protein did not contain any disulfide bonds? If the protein contained a single disulfide bond? Propose an experiment to identify which cysteine residues form the disulfide bond.