biochem 2

Estimate the net charge at pH=7. Assume the pKa values given in the table above.

-1

Calculate to three decimal places the charge on α-melanotropin at pH value of 11.

-2

Estimate the net charge at pH=12. Assume the pKa values given in the table above.

-4

If trypsin cleavage gave two peptides, then where is(are) the S−S bond(s)?

1-3 11-15

To characterize the thrombin in the sample, you must remove two proteins that interfere with the thrombin activity assay: cytochrome c and lactoglobin. You find some CM-cellulose and a phosphate buffer (pH=6.4) on the shelf in your lab. You decide to load the protein sample onto a column of CM-cellulose equilibrated in the pH=6.4 buffer. Predict the order of elution for the three proteins shown in the table below.

1. Lactoglobin 2. Thrombin (wild type) 3. Cytochrome c

average amino acid residue weight in a protein of typical composition

110.76

Calculate the molecular weight of α-melanotropin, using data in the table below.

1646.85 g/mol

Match the following characteristics to α helices, β sheets, or both. 1) 5.4Å/turn A) α helices 2) antiparallel arrangement B) β sheets 3) large dipole moment C) both 4) extensive H-bonding network

1:A; 2:B; 3:A; 4:C

Please match the correct term on the left with the appropriate structural feature of proteins listed on the right. 1) primary structure A) the interaction between two separate protein strands 2) secondary structure B) the sequence of AA 3) tertiary structure C) small sections of organized protein structure, such as helices 4) quaternary structure D) the overall fold of a single protein strand, such as the globulin fold

1:B; 2:C; 3:D; 4:A

Match the amino acid (AA) with its correct side chain category: 1) proline A) negatively charged polar AA 2) histidine B) nonpolar aromatic AA 3) alanine C) positively charged polar AA 4) threonine D) nonpolar AA 5) tryptophan E) polar AA 6) aspartic acid F) nonpolar aliphatic AA

1:D; 2:C; 3:F; 4:E; 5:B; 6:A

1) UCA A) Stop 2) CUA B) Lys 3) AAG C) Start/Met 4) GUA D) Leu 5) AUG E) Val 6) UAG F) Ser

1:F; 2:D; 3:B; 4:E; 5:C; 6:A

Calculate to three decimal places the charge on α-melanotropin at pH value of 5.

2

How many peptides would result if this peptide were treated with (1) cyanogen bromide, or (2) trypsin, or (3) chymotrypsin?

2 none 2

Based on these assumptions, about how many random-coil conformations will be possible for this protein?

2.7⋅10^95

Calculate to three decimal places the charge on α-melanotropin at pH value of 1.

4

If you sketch the titration curve for α-melanotropin (SerTyrSerMetGluHisPheArgTrpGlyLysProVal), near what pH values would you expect the curve to exhibit inflections? Assume the pKas of the N- and C-termini are 7.9 and 3.8, respectively. For side chains, assume the pKa values given.

4,7,9,10,12

Support your answer with the calculation of the pI of the mutant at pH=5.5. Assume that the only factors impacting the change in the pI are the side chains of the mutant protein.

5.15

Calculate the pI (isoelectric point) of α-melanotropin

9

Using the information in table, explain how a point mutation could change a codon for Glu to a codon for Val.

A change of the A in the GAG codon for glutamate to a U would change the codon to GUG, which codes for valine

Which of the following statements about mutations is false?

A knock-out mutation results in a total absence of the mutated protein.

Which of the following statements is NOT true?

A protein stripped of its cofactor or metal ion is known as a holoprotein.

Identify each amino acid shown below in Fischer projection. Indicate whether the D- or L- enantiomer is shown.

A) D-Valine B) D-Threonine C) D-Asparagine

Applications of mass spectrometry include:

A,B, and C

Where is the −S−S- bond (i.e., AB, BC, or AC)?

AC

Part A Part complete Which of the following statements about protein quaternary structure are correct? 1) It involves a complex of two or more proteins interacting with each other. 2) The subunits of the structure can be either identical or different. 3) The interactions between subunits can give rise to indefinite growth of polymeric complexes. 4) Most assemblies of protein subunits have one or more defined axis of rotation.

All of the listed statements are correct.

Which of the following statements about globular proteins are true? 1) The protein folds to make itself as compact as possible. 2) The packing of the protein is such that hydrophilic residues appear on the surface where they can interact with an aqueous environment. 3) Irregularities of the protein's surface allow for the formation of clefts, which are often where the protein promotes a chemical transformation. 4) Regions of secondary structures folding on one another are examples of the protein's tertiary structure.

All of the listed statements are correct.

Identify whether each of the following statements is a likely or an unlikely reason for the failure of the software to determine the native structure.

Almost no: the amino acid sequence alone is not sufficent to specify its native structure

Which statement correctly describes amphipathic (or amphiphilic) helices and sheets?

Amphipathic helices and sheets have predominantly hydrophilic (or hydrophobic) residues on one face.

Part F Part complete If a mutated DNA sequence produces a protein that differs in one central amino acid from the normal protein, which of the following kinds of mutations could have occurred?

An addition mutation and a deletion mutation.

Do you think apamine is synthesized in the form CNCKAPETALCARRCQQH, or is it more likely a product of proteolytic cleavage of a larger peptide? Explain.

Apamine does not have an N-terminal methionine, so at least some proteolytic cleavage must be involved in its synthesis.

Which of the following amino acids would most likely be found on the surface of a protein?

Aspartic Acid and Proline

Which of the following statements is true about a protein that is in an aqueous solution that has a pH equal to the pI of the protein?

At pH = pI, there is no net charge on the protein.

iodoacetate

Cysteine

At pH=6.4, which protein(s) do you predict will remain bound to the column with minimal flow through CM-cellulose?

Cytochrome c

If a DNA sequence is altered from TAGCTGA to TAGTGA, what kind of mutation has occurred?

Deletion

What are the N-terminal and C-terminal residues for the following peptide sequence? Peptide sequence: EASY

E is the N-terminal residue; Y is the C-terminal residue

Which of the following is FALSE when considering the standard genetic code?

Each of the three stop codons can also encode rare modified amino acids.

True or false? A codon is a group of three bases that can specify more than one amino acid.

False

The side chain of ________ has a pKa in the physiological pH range and is therefore often involved in proton transfer during enzymatic catalysis.

Histidine

Which of the following statements regarding the folding of proteins is NOT true?

Hydrophobic residues pack together because the side chains are attracted to each other through weak Van der Waals interactions.

What chromatographic method should make it possible to isolate pure A and B chains?

Ion exchange chromatography. The A chain contains no basic residues in comparison with the B chain, so ion-exchange chromatography should work well

Polyglycine, a simple polypeptide, can form a helix with ϕ=−80∘ , ψ=+150∘. From the Ramachandran plot (see the figure on the left), describe this helix.

It could have polypeptide II helix structure.

Given the helix formed by polyglycine, what can be inferred about its handedness?

It is left-handed.

acetic anhydride

Lysine and N-terminal amine

Find a start signal, and write the amino acid sequence that is coded for. 5'...GCCAUGUUUCCGAGUUAUCCCAAAGAUAAAAAAGAG...3'

MFPSYPKDKKE

Which of the following statements about protein folding and structure are true?

Misfolded proteins often aggregate in large structures in the cell. Chaperones, like the GroEL-ES complex, work by providing a sequestered environment in which proteins can safely explore the conformational space towards productive folding. Misfolded proteins are thermodynamically stable versions of a protein. The native structure of a protein is entirely encoded in its amino acid sequence.

A protein is a large polypeptide. Polypeptides are polymers of amino acids joined together through a special type of bond: the peptide bond. Understanding the chemistry and properties of the peptide bond is fundamental to understanding protein structure (and function). First, identify the N- and C-termini of the peptide. Then, identify those bonds that comprise the peptide bond for each labeled bond. Finally, determine the number of amino acids in the peptide depicted.

N-terminus, G1,G1, not,not, part, part, not, not, C-terminus, with 3 amino acids

Are the helices bound to the DNA likely to be amphiphilic? Explain.

No. DNA is charged and therefore polar, so the DNA-binding helix is likely to be composed of polar residues that interact with either the DNA or the solvent.

The estimate obtained in Part A is surely too large. Give one reason why.

Not all of these conformations will be sterically possible.

Which mutation(s) would not change the remainder of the reading frame of a gene sequence that follows the mutation(s)?

One addition and one deletion mutation.

Glycine and proline are both non-polar amino acids, and both are very likely to be found at the surface of proteins. What physical characteristic of each is responsible for this observation? 1) Both glycine and proline are small. 2) Glycine is small, and proline is rigid. 3) The side chains of both these amino acids make favorable interactions with molecules near the surface of proteins. 4) Both amino acids make strong interactions with each other and therefore are likely to be found next to each other in a protein.

Only statement 2 is correct.

Which of the following characteristics are true about a typical peptide (amide) bond? 1) The bond is planar. 2) There is free rotation about the carbonyl carbon and nitrogen bond. 3) There is substantial double-bond character to this bond. 4) There is a net negative charge on nitrogen and net positive charge on oxygen.

Only statements 1 and 3 are correct.

Which of the following statements regarding Anfinsen's denaturing experiments with ribonuclease A are valid? 1) Exposing the denatured protein to air oxidation and then dialysis to remove urea restored the protein to its original functionality. 2) Removing urea by dialysis and then allowing air oxidation of the denatured protein restored the protein to its original functionality. 3) Denaturing the protein with both urea and β-mercaptoethanol yielded an inactive protein. 4) Protein folding is determined by its primary sequence.

Only statements 2, 3, and 4 are valid.

Which of the following statements about α-keratins is FALSE?

Pairs of α-helices twist about each other in a coiled-coil structure held together entirely by hydrophobic interactions

Give two reasons to explain why a proline residue in the middle of an α-helix is predicted to be destabilizing to the helical structure.

Pro is not able to adopt the ideal ϕ and ψ angles for an α -helix. Pro does not have the α−NH group that acts as a stabilizing H-bond donor in the middle of the helix

thermolysin

SYS,MEHFRWGKP,V

cyanogen bromide

SYSM,EHFRWGKPV

trypsin

SYSMEHFR,WGKPV

The melanocyte-stimulating peptide hormone α-melanotropin has the following sequence: Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val Write the sequence using the one-letter abbreviations.

SYSMEHFRWGKPV

Which of the following modified amino acids is incorporated during translation rather than being modified post-translationally?

Selenocysteine

What is a potential limitation of GFP?

Short wavelengths of excitation light can damage the cell's DNA.

Suggest a method for separating the peptides produced by chymotrypsin treatment

Size exclusion chromatography and Chromatography on a cationic column, at pH=7

Human and sperm whale myoglobin have very similar primary structures. Which of the following statements are correct? 1) The two proteins are very likely related evolutionarily. 2) The differences in the sequences in many instances represent a conservative change (such L for I). 3) The differences in the sequences in many instances represent a nonconservative change (such as D for A). 4) There is no correlation between the two proteins, since they originate from very different species.

Statements 1 and 2 are correct.

The side chain of histidine has a typical pKa value in the range of 6.5-7.4. However, when analyzing the pKa values in a particular protein, scientists determined that one particular His residue has an unusually low pKa value of 4.8. Which of the following statements correctly explain this anomaly? 1) The microenvironment around a residue can impact its pKa value. 2) A positively charged amino acid must be in close proximity to this residue. 3) A negatively charged amino acid must be in close proximity to this residue. 4) This residue must be located on the surface of the protein.

Statements 1 and 2 are correct.

Which statements about β and γ turns are correct? 1) Their purpose is to reverse the direction of the polypeptide chain. 2) There are two types, I and II, which differ mainly in the conformation about the i+1 and i+2 residue amide bond. 3) They typically contain large, hydrophobic residues. 4) Their conformation is held in place through H bonds.

Statements 1, 2, and 4 are correct.

In considering protein secondary structure which of the following is INCORRECT?

The 310 helix is right-handed and often contains proline residues.

What is important in cloning the GFP cDNA into the vector?

The GFP cDNA is in the same reading frame as the target protein.

Where do you predict the N- and C-termini are located for Max?

The N-terminus is interacting with the DNA. The α-amino group of the N-terminus is positively charged and will interact favorably with the negative charge on the phosphodiester backbone of the DNA.

For an amino acid such as aspartic acid, what impact do you expect the two neighboring carboxylic acids to have on the pKa values for each?

The acid with the lower pKa value would increase the pKa value for the other acid.

Which of the following statements about insulin is INCORRECT?

The disulfide bonds form after the final proteolytic cleavage to yield mature insulin.

Which search do you predict will give you alignments with "expect scores" closer to one? Explain your reasoning.

The expect score for the shorter sequence is more likely to be closer to one (i.e., the shorter sequence is more likely to match many entries in the database

Would the chemical modification change the overall charge on the iodoacetate at pH=7?

The negative charge on the peptide will be increased

Would the chemical modification change the overall charge on the ATP-dependent kinases at pH=7?

The negative charge on the peptide will be increased.

Is the pI of the mutant protein predicted to be greater than, less than, or the same as the pI of the normal protein?

The pI of the mutant protein predicted to be less than the pI of the normal protein

Do you expect the pI for the sickle-cell β-globin to be higher or lower than the pI for wild-type β-globin? Explain.

The pI of the sickle-cell globin will be higher than the wild-type globin because the mutation replaces a negatively charged side chain with one that carries no charge.

Would the chemical modification change the overall charge on the acetic anhydride at pH=7?

The positive charge on the peptide will be reduced

A short 8-residue sequence of a polypeptide is determined to have φ angles ranging from -65 degrees to -80 degrees and ψ angles ranging from -40 degrees to -50 degrees. What conclusion can be drawn from this data?

This segment has helical content.

If the sequence ATGCATGTCAATTGA were mutated such that a base were inserted after the first G and the third T were deleted, how many amino acids would be changed in the mutant protein?

Two

If apamine does not react with iodoacetate, then how many disulfide bonds are present?

Two disulfides.

kinase + ATP

Tyrosine and Serine and Threonine

You are surprised to observe that the patient's thrombin flows through the CM-cellulose column at pH=6.4 faster than expected based on its pI. Confident in your technique, you suspect the patient's thrombin is different from wild-type thrombin. Using a different buffer system, you manage to purify some of the patient's thrombin and you submit the purified sample for amino acid sequencing. The sequence analysis shows that the patient's thrombin contains a mutation in the enzyme active site. A lysine residue in the wild type has been mutated to an asparagine in the patient's thrombin. Does this mutation explain the anomalous CM-cellulose binding behavior you observed?

Yes. Loss of a (+)-charged side chain will increase the (−) charge density on the protein, making it less likely to bind to the CM cellulose.

At pH=0, the net charge on a polypeptide will be negative.

false

Conservative amino acid changes never affect stability or function of a protein.

false

Most proteins have blocked amino and carboxyl terminals.

false

When referring to the amino acid sequences of proteins, sequence homology is the same as sequence similarity.

false

We can use an energy landscape funnel as a way to visualize the various pathways a given amino acid sequence may take to achieve its final folded state.

folding, native, fully unfolded, misfolded

For which of the following may GFP fusion technology NOT be a good technique to use?

for target proteins whose structure is altered by a GFP fusion

The Foundation Figure began by discussing the importance and relevance of protein structure. Fill in the blanks in the paragraph below with a word or phrase from the word bank that best completes the sentence.

function, shape, hydrogen bonding, complementary to

All amino acids have a chiral α-carbon EXCEPT ________.

glycine

________ between amide protons and carbonyl oxygens is necessary to stabilize a regular folding of protein secondary structure.

hydrogen bonding

List two different ways you could change the buffer to elute the bound protein(s) and achieve proper separation of the proteins.

increasing the ionic strength of the buffer and add a buffer with pH of 8

GFP fusion technology would be best used for which of the following experiments?

localizing the compartment where Golgi proteins are found

What is the net charge on the following peptide at pH = 0? Peptide sequence: DSVK

net charge = +2

If so, is the pI of the modified peptide higher or lower than that of the untreated peptide?

pI is lower in every case.

A ________ plot describes which structures in a polypeptide are sterically possible and which are not based on the angles of rotation about the backbone Namide -Cα and Cα-Ccarbonyl bonds.

ramachandran

Where in a globular protein is the amino acid alanine likely to be located?

the hydrophobic interior

In size exclusion chromatography, the smallest proteins are eluted last.

true

Protein biosynthesis uses only L-amino acids.

true

The amino acid side chain residues in an α helix point outwards away from the center of the helix.

true

Cells expressing the GFP fusion protein will show florescence __________

where the GFP fusion protein is localized in the cell